Multilevel Regulation of IL-6R by IL-6CsIL-6R Fusion Protein According to the Pr - 《stemcells》干细胞学杂志干细胞之家



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作者：David Camparda, Marc Vassea, Stefan Rose-Johnb, Florent Poyera, Marek Lamacza, Jean-Pierre Vanniera作者单位：a Laboratoire Micro-Environnement et le Renouvellement Cellulaire Intgr (EA ), Facult de Mdecine-Pharmacie de Rouen, Rouen, France;b Department of Biochemistry, Christian-Albrechts-Universitt zu Kiel, Kiel, Germany : [7 o4 H9 U& c7 Y/ E






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      【摘要】
   Interleukin-6 (IL-6) and its soluble receptor (sIL-6R) are major factors for maintenance and expansion of hematopoietic stem cells (HSCs). Sensitivity of HSCs to IL-6 has been previously studied, in part by measuring the expression of IL-6R on the membrane (mIL-6R). Several studies have described the regulation of cell surface expression of IL-6R by several cytokines, but the role of glycoprotein 130 activation has not yet been investigated. In this study, CD133  cells were purified from adult peripheral blood and were precultured in the absence or presence of 5-fluorouracil (5-FU) for selection of quiescent HSCs. Cells were cultured with continuous or pulsed stimulations of an IL-6 ¨CsIL-6R fusion protein (hyperinterleukin-6 ) to 1) detect mIL-6R by flow cytometry, 2) assess mIL-6R and sIL-6R RNAs by reverse transcription-polymerase chain reaction, 3) measure sIL-6R in supernatants by enzyme-linked immunosorbent assay, 4) analyze cell-cycle status, and 5) perform long-term culture-initiating cell assays. The level of mIL-6R¨C cells was preserved by 5-FU incubation. HIL-6 increased steady-state mIL-6R RNA and expression rate on HSCs, independently of treatment with 5-FU. Enhanced production of sIL-6R was observed with short pulses of HIL-6 on CD133  5-FU-pretreated cells. This overproduction of sIL-6R was abrogated by tumor necrosis factor- protease inhibitor-1, an inhibitor of a disintegrin and metalloprotease proteases, suggesting the shedding of mIL-6R. This phenomenon was mediated through the phosphatidylinositol-3'-kinase pathway and was involved in the maintenance of primitive HSCs. In conclusion, expression and production of IL-6R are tightly regulated and stage specific. We assume that sIL-6R produced by shedding should be involved in autocrine and paracrine loops in the HSC microenvironment. % G& K$ X! L! [' ~, n% y! A* h
      【关键词】 CD  cells Hyperinterleukin- IL-R ADAM proteases -Fluorouracil+ j% N. q% ^  D1 l  _3 r- T! c
   INTRODUCTION6 D; f) r! K- Q9 f' {
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Over the last few years, numerous studies have suggested that the stimulation of glycoprotein 130 (gp130) is required for maintenance of hematopoietic stem cells (HSCs) .7 f% k4 Q, f0 w( a

CD34  cells do not express and secrete IL-6 , but until now there has been no report devoted to the regulation of mIL-6R and sIL-6R on HSCs by gp130 activation. In the present study, we demonstrate that gp130 activation by HIL-6 stimulates mIL-6R expression and sIL-6R production through a post-translational mechanism involving a disintegrin and metalloprotease (ADAM) protease.
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MATERIALS AND METHODS

CD133  Cell Purification( n  h! `/ U) l4 w3 n
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PB cells were obtained from normal volunteers after informed consent. One hundred milliliters of blood was collected on 15 ml of ACD-solution (38 mM citric acid monohydrate, 74.5 mM trisodium citrate, 136 mM D-glucose), then diluted 1:1 in RPMI 1640 medium (Eurobio, Courtaboeuf, France, http://www.eurobio.fr) and centrifuged for 10 minutes at 800 rpm to remove the platelet-rich-plasma phase. Mononuclear cells (MNCs) were isolated using Ficoll-Hypaque (1.077 g/ml) (Eurobio) density centrifugation. Depletion of monocytes was performed by plastic adherence for 45 minutes at 37¡ãC and 5% CO2 in RPMI 1640.2 h  B2 `4 |5 N
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The CD133  MNC fraction was isolated with microbeads selection using a Vario magnetic affinity cell sorting (MACS) separator and MS separation columns (Miltenyi Biotec, Paris, http://www.miltenyibiotec.com). This isolation was performed by positive selection of CD133-expressing cells. The CD133  cells were suspended in phosphate-buffered solution (PBS; Eurobio, France), 0.1% bovine serum albumin (Sigma-Aldrich, Lyon, France, http://www.sigmaaldrich.com), and 2 mM EDTA (Sigma-Aldrich) buffer. They were directly labeled by incubation for 30 minutes at 4¡ãC with a monoclonal antibody (clone AC133, epitope 1) coupled to microbeads. Cells were washed in separation buffer and laid on an MS column placed in a magnetic field. The magnetically labeled CD133  cells are retained on the column, while unlabelled CD133¨C cells pass through. After removal of the column from the magnetic field, the magnetically retained CD133  cells can be eluted as the positively selected fraction. Living cells are counted on a Thoma slide using trypan blue exclusion.+ b- x, Q! ]- M  h# E3 n
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5-FU Treatment of Cells

Freshly isolated CD133  cells were cultured in a 25-cm2 flask in 5 ml of Iscove¡¯s modified Dulbecco¡¯s medium (IMDM) (Gibco-BRL, Cergy Pontoise, France, http://www.invitrogen.com) supplemented with 10% fetal calf serum (FCS; Eurobio), 1% penicillin and streptomycin (50 µg/ml; Gibco-BRL), 10 ng/ml IL-3 (R&D Systems, Lille, France, http://www.rndsystems.com), and 25 µg/ml 5-fluorouracil (5-FU) (Merck, Lyon, France, http://www.merck.fr). After 24 hours of incubation at 37¡ãC and 5% CO2, cells were washed three times with IMDM, and the number of living cells was evaluated by staining with trypan blue.- `5 U$ s* v( f% }7 r

Recombinant Proteins, Neutralizing Antibody, and Inhibitors8 V2 N3 P" W; r# L
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The following recombinant purified human cytokines used in this study were purchased from R&D Systems: stem cell factor (SCF), thrombopoietin (Tpo), Flt-3 ligand (Flt-3), IL-3, IL-6, erythropoietin (Epo), and G-CSF. Endogenous transforming growth factor (TGF)-ß1 was neutralized by an anti-human TGF-ß1 monoclonal antibody (clone 9061) (R&D Systems). 4ß-Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma-Aldrich. Tumor necrosis factor- protease inhibitor-1 (TAPI-1), an ADAM inhibitor, LY294002, an inhibitor of phosphatidylinositol-3'-kinase (PI3K), and U0126, an inhibitor of mitogen-activated protein kinase (MAPK)/mitogen-activated protein kinase kinase (MEK)-1 and MEK-2, were purchased from Calbiochem (VWR International S.A.S., Fontenay sous Bois, France, http://www.merckbiosciences.co.uk).

A high concentration of sIL-6R is required to stimulate gp130-expressing cells because the IL-6¨CsIL-6R complex is unstable. So, a fusion protein consisting of IL-6 and sIL-6R covalently linked by a flexible polypeptide was used to stimulate HSCs. This fusion protein, called hyperinterleukin-6 (HIL-6) has been shown to be useful at 100- to 1,000-fold lower concentrations than unlinked IL-6 and sIL-6R .
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Liquid Cultures$ o( a! e/ T8 \8 j

Liquid cultures were established with freshly isolated or 5-FU-treated CD133  cells from PB in 96-well plates in serum-free survival medium (Stemspan SFT) composed of Stemspan (StemCell Technologies Inc., Grenoble, France, http://www.stemcell.com) supplemented with SCF (50 ng/ml), Flt-3 (50 ng/ml), and Tpo (10 ng/ml). Cells were plated at a density of 2.0¨C3.0 x 105 cells/well. Cells were incubated at 37¡ãC and 5% CO2 with 10 µg/ml anti-TGF-ß1 for 48 hours. Supernatants were gently recovered for enzyme-linked immunosorbent assay (ELISA), and cells were harvested for reverse transcription-polymerase chain reaction (RT-PCR) analysis. HIL-6 stimulations were carried out according to the following tests. Cells were exposed to 100 ng/ml HIL-6 for 2 or 24 hours and submitted directly to the analysis of IL-6R mRNA expression. Because HIL-6 is recognized by the anti-sIL-6R antibody of the ELISA kit, cells were stimulated with HIL-6 for 2 or 24 hours, and the media were removed and replaced with new HIL-6-free survival medium. These "pulsed" cells were also analyzed for IL-6R mRNA expression.
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For mIL-6R detection by flow cytometry, freshly isolated CD133  cells were seeded in 96-well plates at a density of between 7.5 x 104 and 105 cells per well, in 200 µl of Stemspan SFT. Cells were incubated for 24 or 48 hours at 37¡ãC and 5% CO2 in the survival medium, with or without 5-FU (250 µ g/ml), and with HIL-6 (100 ng/ml) and/or anti-TGF-ß1 (10 µg/ml).: D- O; C/ z/ z( f( e
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Limiting Dilution Analysis of Long-Term Culture-Initiating Cells/ Y& T# B* ?" L! `1 ~( e
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Cocultures were established by incubating sorted CD133  cells in 96-well plates precoated with the murine stromal cell line MS-5 (DSMZ, Braunschweig, Germany, http://www.dsmz.de) . The MS-5 stromal cell line was maintained in -modified Eagle¡¯s medium (-MEM) (Eurobio) supplemented with 10% FCS, 2 mM glutamine (Gibco-BRL), 2 mM pyruvate, and 1% penicillin and streptomycin (50 µg/ml). After a 2-week culture, MS-5 cells were irradiated (2 x 25 Gy; ISP Technologies Inc., Anaheim, CA, http://www.isptechinc.com) and seeded in 96-flat-bottom-well plates at 3.5 x 104 cells per well. The next day, the sorted CD133  cells were plated at six different dilutions (range, 25¨C800 cells/well). Twenty-four replicates were done for each dilution. Four different conditions were assessed: control culture in 200 µl of Stemspan, Stemspan supplemented with 200 µM TAPI, Stemspan supplemented with 100 ng/ml HIL-6, and Stemspan supplemented with a combination of TAPI and HIL-6. Plates were maintained at 37¡ãC in a fully humidified atmosphere of 5% CO2 in air, and cultures were fed weekly by a 100-µl Stemspan change.

After 5 weeks, plates were washed and cells were directly plated in a semisolid collagen medium to determine the clonogenic cell content of each long-term culture (LTC). In brief, supernatants were removed and cells were plated in 86 µl of collagen and 120 µl of Megacult (StemCell Technologies) supplemented with 50 ng/ml SCF, 10 ng/ml IL-3, 10 ng/ml IL-6, 5 ng/ml G-CSF, and 1 U/ml Epo. After 14 days of culture at 37¡ãC in a fully humidified atmosphere of 5% CO2 in air, colonies were counted: a well was scored as positive if at least one burst-forming unit erythroid (BFU-E), colony-forming unit granulocyte-macrophage (CFU-GM), or colony-forming unit granulocyte erythroid-megakaryocyte macrophage (CFU-GEMM) was detected. The LTC-initiating cell (IC) frequency for each condition was calculated from the proportion of negative wells (no CFU present) and the method of maximum likelihood .4 i; h7 B2 G1 i* f8 l7 N% E6 ~

mIL-6R Detection by Flow Cytometry1 X  N* M  O2 U: b' I$ K
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Cultured CD133  cells were harvested and the wells were washed three times with PBS. Cells were suspended in 100 µl of cold PBS and incubated at 4¡ãC for 30 minutes with 20 µl of biotinylated mouse anti-human IL-6 receptor ( chain) mono-clonal antibody (clone M5) (BD Pharmingen, Le Pont de Claix, France, http://www.bd.com). Then, cells were washed and suspended in 100 µl of cold PBS and incubated in the dark at 4¡ãC and for 30 minutes with 10 µl of streptavidin-phycoerythrin (PE) conjugate (1:50) (BD Pharmingen). After a last wash, cells were suspended in 300 µl of PBS and all samples were analyzed on EPICS XL-MCL flow cytometer (Beckman Coulter, Roissy CDG, France, http://www.beckmancoulter.com). Data were expressed as the percentage of positive cells.4 R' J/ K2 ~. u) m+ ~1 O
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Negative controls included unstained cells and cells only stained with phycoerythrin (PE)-conjugated isotype control IgG (Immunotech, France) or only with the streptavidin-PE. As cells incubated with only streptavidin-PE have shown the strongest non-specific staining, they have been routinely used for control.

Cell-Cycle Analysis+ H. n7 \6 T7 i# X( S$ F9 J; n
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Cultured CD133  cells were harvested and washed in cold PBS. Cells were resuspended in 1 ml PBS, 0.5% FCS, and 5% DMEM and fixed with cold (at ¨C20¡ãC) ethanol 95%. Cells were washed and stained with 0.5 ml of 50 µg/ml propidium iodide (PI; Sigma-Aldrich-Aldrich, France) supplemented with 100 µg/ml RNase A (Amersham Bioscience, France). Samples were incubated 20 minutes in the dark at room temperature, and were finally analyzed on EPICS XL-MCL flow cytometer (Coulter, France).
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RT-PCR for Detection of Alternatively Spliced and Nonspliced IL-6R mRNA( k0 l9 U* I  e. ?2 r
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The expression of IL-6R mRNA was investigated using RT-PCR. Cultured cells were slowly centrifuged, and supernatants were removed and kept for ELISAs. Wells containing CD133  cells were washed three times with PBS, and final cell suspensions were collected and centrifuged. Total cellular RNA was isolated using SV Total RNA Isolation System (Promega, Charbonni¨¨res, France, http://www.promega.com) according to the manufacturer¡¯s instructions. This device permits the extraction of pure RNA from a small number of cells using DNase treatment and immediate inactivation of endogenous RNases.* ~8 [, J" R4 W. V* T: ]

The oligonucleotide primers for IL-6R were selected according to the sequence data published by Horiuchi et al. . The primer sites flanked the transmembrane domain of the receptor. The IL-6R sense primer was 5'-ACGCCTTGGACA-GAATCCAG-3' and the antisense primer was 5'-TGGCTC-GAGGTATTGTCAGA-3'. By using this primer set, it is possible to distinguish between the two different-sized IL-6R mRNA templates, that is, the mRNA coding for mIL-6R and the alternatively spliced mRNA that lacks the transmembrane sequence and codes for sIL-6R. In this way, amplifications products of 398 and 304 bp were formed from the nonspliced and spliced mRNA, respectively.

ß-Actin serves as the internal control for RT-PCR. Its sense primer was 5'-ATCTGGCACCACACCTTCTA-3' and the antisense primer was 5'-CTCGGTGAGGATCTTCATGA-3' and generated an amplification product of 333 bp.
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Five microliters of total RNA for one sample was denatured at 94¡ãC for 2 minutes. RNA template was added to the RT-PCR reaction mixture containing 10 µl of 1x AMV/Tfl reaction buffer, 1 mM MgSO4, 0.2 mM dNTP mixture, 5 U of Tfl DNA polymerase, 5 U of AMV reverse transcriptase (AcessQuick RT-PCR System; Promega), 160 pM of sense and antisense primers (Proligo, Hamburg, Germany, http://www.proligo.com), and nuclease-free water adjusted to a final volume of 50 µl. The reverse transcription reaction was performed at 48¡ãC for 45 minutes. The thermal profile for IL-6R PCR involved denaturation at 94¡ãC for 30 seconds, primer annealing at 55¡ãC for 1 minute, and extension at 68¡ãC for 1 minute. Forty cycles were performed using a 480 Thermal Cycler (Perkin-Elmer Cetus, Courtaboeuf, France, http://www.perkinelmer.fr). The thermal profile for ß-actin was repeated for 30 cycles with denaturation at 94¡ãC for 30 seconds, annealing at 60¡ãC for 30 seconds, and extension at 68¡ãC for 30 seconds. Final extensions for both IL-6R and ß-actin PCRs were performed at 68¡ãC for 7 minutes before soaking of the amplified PCR products at ¨C 20¡ãC.* C! S& U7 Z: c' f
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The amplified PCR products were electrophoresed for 45 minutes at 95 V through a 2.0% agarose (BioWhittaker Molecular Applications, Rockland, ME, http://www.bmaproducts.com) gel containing 0.5 µg/ml ethidium bromide. A 100-pb DNA Ladder (Promega) was used as a molecular weight marker. The gel was visualized under UV light.- V7 ]3 n: ^3 g6 K2 P. @4 ?3 L! _

Estimation of band intensity was performed on the gel analyzer module of the ImageJ 1.32j program (Public Domain, National Institutes of Health, Bethesda, MD, http://www.nih.gov). Intensities of the IL-6R' bands for a sample were normalized according to the respective intensity of the ß-actin band. Then, the normalized IL-6R' bands for the samples of stimulated cells were expressed according to the normalized IL-6R' bands for the samples of cells in control conditions.# \2 {! f5 \% |+ ~% O' J3 E9 ~7 K
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Measurement of sIL-6R from Cell Culture Media

Cultured cells were slowly centrifuged, and supernatants were collected and centrifuged to eliminate potentially contaminating cells. Samples were kept frozen at ¨C20¡ãC for further analysis. The levels of sIL-6R in culture media were determined using the Quantikine ELISA kit for human sIL-6R (R&D Systems) according manufacturer¡¯s instructions. Absorbance values were measured at 450 nm.

Statistical Analysis$ s/ j. E, [+ u9 }6 }
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Statistical analyses were performed using S-PLUS 2000 Software (MathSoft; Engineering and Education, Inc., Cambridge, MA). The probability of a significant difference between groups was determined using the Mann-Whitney U test for nonpaired data. Analysis of the cell-cycle assay was performed under Prism 4 (GraphPad Software, Inc., San Diego, CA) with a one-way analysis of variance followed by Tukey¡¯s multiple comparison test. Statistical analysis of the limiting dilution analysis was performed using L-Calc software (StemCell Technologies Inc.).
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RESULTS
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HIL-6 Enhances the Expression of mIL-6R in CD133  Cells

Untreated or 5-FU-pretreated CD133  cells were cultured for 2 or 24 hours in Stemspan SFT, with or without HIL-6, in conditions of continuous or pulsed stimulations. Cells were collected and total RNA was extracted for RT-PCR of IL-6R RNA. Amplicons were electrophoresed (Fig. 1A) and semiquantified by analysis of band intensity (Fig. 1B). The soluble spliced form of IL-6R was less expressed than the membrane-bound IL-6R form and was even undetected in some donor samples (data not shown).2 d& D9 P8 h6 V) i

Figure 1. mIL-6R and sIL-6R transcripts after HIL-6 exposition. Reverse transcription-polymerase chain reaction was performed for IL-6R and ß-actin RNA, and amplicons were electrophoresed on a 2% agarose gel. (A): A representative experiment with 5-FU-pretreated cells after a 2-hour pulse of 100 ng/ml HIL-6. Intensities of mIL-6R (398 bp) and sIL-6R (304 bp) bands were measured using ImageJ Software. Intensity values (arbitrary units) of each sample were normalized according to total RNA content, evaluated by intensity of the respective ß-actin band. Expression of mIL-6R and sIL-6R for the cells stimulated by HIL-6 (black bars) were normalized according those of the respective control (white bars). (B): Same representative experiment. IL-6R RNA was evaluated in a series of HIL-6-stimulated and unstimulated and 5-FU-pretreated and untreated CD133  cells. (C): To evaluate the time-dependent biological effects of HIL-6, cells were cultured for 2 or 24 hours with HIL-6 then lysed immediately (continuous stimulation) or cultured for an additional 24-hour period in HIL-6-free medium before lysis (pulsed stimulation). Results are mean ¡À standard error of the mean (n = 4). *, p
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Continuous stimulations for 2 or 24 hours with HIL-6 did not modify the mIL-6R expression on untreated- and 5-FU-pretreated CD133  cells (Fig. 1C). However, significantly greater mIL-6R RNA was observed in CD133  cells when they were stimulated for 2 or 24 hours with HIL-6 then cultured for an additional 24-hour period in HIL-6-free medium. The pulsed stimulations for 2 and 24 hours also resulted in greater sIL-6R RNA expression, in and only in 5-FU-untreated CD133  cells. No stimulating effect of HIL-6 on sIL-6R RNA was observed in 5-FU-pretreated CD133  cells. Transcripts of sIL-6R were decreased by continuous and pulsed stimulations of 2 hours with HIL-6. Longer stimulations for 24 hours did not modify the expression of sIL-6R on 5-FU-pretreated CD133  cells.8 i, T. u7 V7 M: k+ |' K# C' Z' J
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HIL-6 Increases sIL-6R Production on 5-FU-Pretreated CD133  Cells2 Y" d. z; m7 l9 ^# M

Similarly to another study , HSCs constitutively produced sIL-6R at a basal level (54.8 ¡À 6.5 pg/ml per 1.5 x 105 CD133  untreated cells for a period of 24 hours) (Fig. 2). 5-FU pretreatement did not significantly modify the basal production of sIL-6R (37.7 ¡À 4.6 pg/ml after 24 hours).
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Figure 2. Transient and short exposure to HIL-6 increases production of sIL-6R by 5-fluorouracil (5-FU)-pretreated CD133  cells. Concentrations of sIL-6R in supernatants of untreated (white bars) or 5-FU-pre-treated (black bars) CD133  cells (1.5 x 105 per well). Cells were incubated for 2 or 24 hours with 100 ng/ml HIL-6, and media were replaced with new HIL-6-free medium for 24 hours. Supernatants were collected and used for determination of sIL-6R concentration by enzyme-linked immunosorbent assay. Results are mean ¡À standard error of the mean (n = 5¨C12). ***, p
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HIL-6 was recognized by IL-6R antibody provided in the ELISA kit so it was not possible to evaluate de novo sIL-6R production in the supernatant of cultures stimulated by HIL-6. However, we evaluated the sIL-6R production after a pulse of HIL-6 for 2 or 24 hours and an additional culture for 24 hours without the fusion protein. In such experiments, 5-FU-untreated CD133  cells stimulated with HIL-6 pulses did not significantly modify their production of sIL-6 (Fig. 2). However, the sIL-6R production by 5-FU-pretreated CD133  cells was 15-fold greater after HIL-6 pulses for 2 hours. A more prolonged exposure of 5-FU-pretreated CD133  cells to HIL-6 seemed to stimulate sIL-6R production, but this was not significant.

Effect of IL-11 and LIF on IL-6R Expression on 5-FU-Treated CD133  Cells

The effect of other cytokines belonging to the IL-6 family was tested on the expression of the IL-6R gene and sIL-6R production. After 5-FU treatment, CD133  cells were exposed to IL-11 (100 ng/ml) or LIF (100 ng/ml) for 2 or 24 hours or pulsed for 2 hours with these same cytokines. IL-11 had no effect on IL-6R transcripts, except on sIL-6R mRNA when cells were cultured for 24 hours with this cytokine (Fig. 3A). The level of sIL-6R mRNA was significantly greater (p
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Figure 3. mIL-6R and sIL-6R expression after IL-11 and LIF exposure. (A): Normalized expression of mIL-6R and sIL-6R mRNA in 5-FU-pretreated CD133  cells stimulated or not with 100 ng/ml IL-11 or 100 ng/ml LIF. Cells were cultured for 2 or 24 hours with IL-11 or LIF and then lysed immediately (continuous stimulation) or cultured for an additional 24-hour period in IL-11 and LIF-free medium before lysis (pulsed stimulation). Results are mean ¡À standard error of the mean (SEM) (n = 4¨C5). *, p

LIF exposure resulted in a significantly greater level of mIL-6R mRNA for each type of stimulation used (Fig. 3A). This small upregulation slightly enhanced the expression of mIL-6R on 5-FU-pretreated CD133  cells (Fig. 3B). The effect of a 2-hour pulse with LIF was minor in regard to continuous stimulations for 2 hours and 24 hours. Neither sIL-6R mRNA level nor sIL-6R production was significantly greater after LIF stimulation (Fig. 3A, 3C).
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mIL-6R Is Less Expressed on 5-FU-Resistant CD133  Cells
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To document the potential effects of HIL-6 on the regulation of mIL-6R, we first measured the mIL-6R expression rate on surviving untreated and 5-FU-treated CD133  cells in Stem-span SFT for 24 or 48 hours. After a 24-hour culture, 44.3% ¡À 6.3% of untreated (Fig. 4, n = 7) and 39.8% ¡À 4.7% of 5-FU-treated CD133  cells (n = 5) expressed mIL-6R without any significant difference. However, 5-FU-untreated cells expressed significantly (p ; J; d2 ^. ^, N8 q
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Figure 4. Expression of mIL-6R is lower on 5-fluorouracil (5-FU)-resistant CD133  cells. CD133  cells were cultured for 24 or 48 hours in Stemspan SFT without (white bars) or with 5-FU (black bars) and were collected for determination of mIL-6R expression by flow cytometry. Only living cells were gated. After a 24-hour period of culture, no significant difference was observed between 5-FU-treated and untreated cells, but after a 48-hour period of culture, the mIL-6R expression rate was lower in 5-FU-resistant cells. Results are mean ¡À the standard error of the mean (n = 5¨C7). *, p 2 O4 @4 l/ {) K6 r; Q
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Forward scatter/side scatter analysis yielded three subpopulations in the whole CD133  population (Fig. 5A). The most numerous subpopulation, delimited by gate A in Figure 5A, was composed of small living cells. On the diagram, dead cells characterized by a smaller diameter and a high autofluorescence in the red-light spectrum, were localized just below the small living cells. Large living cells were delimited in gate E (Fig. 5A). They were identified on May-G¨¹nwald Giemsa (MGG)-stained cytospins and characterized by a large nucleus (Fig. 5E). After culture with 5-FU, this subpopulation failed to be observed both on MGG staining (Fig. 5D, 5F) and the cytometry diagram. Interestingly, more dead cells were counted (gate E, Fig. 5D). These results suggest that large 5-FU-sensitive cells may be dividing cells emerging from the initial subpopulation of CD133  living cells after a period of culture.
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Figure 5. Emergence from untreated CD133  cells of a subpopulation with greater mIL-6R expression. Untreated (A) and 5-FU-treated (D) CD133  cells from each donor were cultured in Stemspan SFT for 24 hours and were harvested for determination of mIL-6R expression by flow cytometry (a representative experiment is shown). The original CD133  population was gated, gate A (A), and its mIL-6R expression rate was determined (C). In the absence of 5-fluorouracil (5-FU) treatment, a particular subpopulation was observed after a 24-hour period of culture, (A) gate E compared with (D). Large cells (E) (black arrows) were only observed on May-G¨¹nwald Giemsa¨Cstained cytospins prepared from untreated cells (compared with 5-FU-treated cells (F). The mIL-6R expression rate (B) for this subpopulation was higher than for the CD133  initial cells (G). Results are mean ¡À standard error of the mean (n = 3).*, p , K& M) o% b$ V& M6 l) y% F

In addition, these large 5-FU-sensitive cells exhibited a greater expression of mIL-6R (73.4% ¡À 5.6%; Fig. 5G) than the initial population of CD133  cells (44.3% ¡À 6.4%).7 u1 {- p2 F+ {$ ]' v* f; K
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HIL-6 Does Not Enhance Recruitment of CD133  Cells in Cell Cycle2 J) d( ~' t$ z( D, J, M- C
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Cell-cycle status of CD133  cells was determined in 5-FU-pretreated and untreated cells. Sorted cells were pretreated or not with 5-FU for 24 hours then cultured in Stemspan SFT, with or without HIL-6, for 24 or 48 hours. As already described , most CD133  cells are in the G0 phase. The rate of cells in the G0 and pre-G0 (necrotic/late apoptotic cells) phases remained constant over time when no 5-FU pretreatment was applied (Fig 6B). In contrast, the number of 5-FU-pretreated cells in the pre-G0 phase was significantly greater after 48 hours in culture (Fig. 6A). Consequently, significantly fewer cells in the G0 phase were observed in 5-FU-pretreated cells cultured for 24 hours and more were observed in cells cultured for 48 hours (Fig 6B). The 5-FU-induced death of cycling cells was confirmed by analysis of the S phase. As expected, the number of cells in the S phase was lower in 5-FU-treated than in untreated cells (Fig 6C). Nevertheless, differences between 5-FU-pretreated and untreated cells in G2/M were not significant (Fig. 6D). Finally, HIL-6 did not modify the frequency of cells in any phase of the cell cycle, whatever the conditions of culture (5-FU pretreatment and duration of culture).
9 Z* G1 h6 j: ~3 }2 `' p' R/ v  p5 i! V
Figure 6. Effect of 5-fluorouracil (5-FU) treatment and HIL-6 on cell-cycle status of CD133  cells. Untreated (white bars) and 5-FU-pretreated (black bars) CD133  cells were cultured in Stemspan SFT alone or with 100 ng/ml HIL-6 for 24 or 48 hours. Cells were harvested, and their distribution in the pre-G0 (A), G0/G1 (B), S (C), and G2/M (D) phases was measured by propidium iodide staining. Results are mean ¡À standard error of the mean (n = 5). *, .01  p
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HIL-6 Increases mIL-6R Expression on Untreated and 5-FU-Resistant CD133  Cells# }' f. i2 q0 q- e1 K

As shown by flow cytometry experiments, HIL-6 produced a greater mIL-6R expression rate on 5-FU-treated and untreated CD133  cells (Fig. 7). This upregulation was observed after a 24-hour stimulation and was sustained until 48 hours in untreated cells (Fig. 7A). The addition of anti-TGF-ß1 to HIL-6 did not modify the HIL-6-induced greater mIL-6R expression rate.

Figure 7. mIL-6R expression on untreated or 5-FU-treated CD133  cells exposed to hyperinterleukin-6 (HIL-6) and/or anti-transforming growth factor (TGF)-ß 1. Untreated (A) or 5-FU-treated (B) CD133  cells were cultured in Stemspan SFT alone (white plain) and with 100 ng/ml HIL-6 (white hatched), 10 µg/ml anti-TGF-ß1 monoclonal antibody (grey plain), or with a combination of both HIL-6 and anti-TGF-ß1 (grey hatched). After a 24- or 48-hour period of culture, cells were harvested and stained for mIL-6R detection by flow cytometry. Results are mean ¡À the standard error of the mean (n = 5) *, .01  p 3 u9 n3 r2 G, h7 ^
  v/ W) Q4 N8 @
The data concerning the effect of HIL-6 on 5-FU-resistant HSCs appeared to be more complex. The mIL-6R expression rate was significantly greater after a culture of 48 hours with HIL-6 (Fig. 7B). In contrast, this was not observed after a shorter period of culture (24 hours). However, the HIL-6-induced greater mIL-6R expression was restored in the presence of an anti-TGF-ß1 antibody (Fig. 7B). A weak downregulation of mIL-6R was observed when 5-FU-resistant CD133  cells were cultured with anti-TGF-ß1 for 24 hours.

Activation of ADAM Proteases by PMA and HIL-6 Increases sIL-6R Production by CD133  Cells2 A. N& n$ s  `4 h4 j1 [" C! y
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The metalloproteinase and disintegrin ADAM-17 is known to be involved in the shedding of mIL-6R . We studied the role of proteolytic cleavage of mIL-6R using TAPI, an inhibitor of ADAM proteases. In a set of experiments, 5-FU-pretreated or untreated CD133  cells were seeded at 1.5 x 105 per well in Stemspan SFT alone, with 10¨C7 M PMA, with 200 µM TAPI, or with a combination of PMA and TAPI. After a 4-hour culture, the mIL-6R expression rate, mean fluorescence intensity (MFI), and sIL-6R level in the supernatant were measured (Fig. 8).! N' p! t* i; Q" z' I- e
9 ?- j6 s. n6 |5 i. [6 i: q
Figure 8. Shedding of mIL-6R by a disintegrin and metalloprotease (ADAM) proteases in CD133  cells. Untreated (white bars) or 5-flouracil-pretreated (black bars) CD133  cells (1.5 x 105 cells per well) were cultured for 4 hours in Stemspan SFT alone and with 10¨C7M PMA, 200 µM TAPI, or a combination of PMA and TAPI. After this period of culture, cells were harvested for determination of mIL-6R expression rate (A) and mean fluorescence intensity (B) by flow cytometry. These results are normalized according to the control values. Supernatants were collected and used for determination of sIL-6R concentration by enzyme-linked immunosorbent assay (C). Results are mean ¡À the standard error of the mean (n = 5¨C12). *, .01  p
& W7 ^4 t) H! Z5 P9 f
In CD133  cells, PMA induced a lower mIL-6R expression rate (Fig. 8A). Dramatically fewer mIL-6R molecules were detected on each CD133  cell, independently of 5-FU pretreatment (Fig. 8B). In the absence of PMA, TAPI did not significantly modify basal mIL-6R expression on untreated cells, but produced a slightly lower expression on 5-FU-pretreated CD133  cells. Incubation with PMA and TAPI restored mIL-6R expression to the control level, suggesting that TAPI may prevent PMA-induced shedding by ADAM-17 and particularly on 5-FU-treated cells (Fig. 8A). This effect was less obvious in untreated cells (Fig. 8B). In parallel, ELISA experiments show a significantly greater sIL-6R concentration in supernatants of 5-FU-pretreated CD133  cells stimulated with PMA (Fig. 8C). TAPI inhibited the release of mIL-6R in the extracellular medium. In untreated CD133  cells, the PMA-induced greater sIL-6R concentration in supernatants was not significant.

The direct evidence that HIL-6 induced activation of ADAM proteases was brought by the measurement of the sIL-6R concentration in supernatants of 5-FU-pretreated CD133  cells pulsed for 2 hours with HIL-6 in the presence of TAPI (Fig. 9A). The inhibitor of ADAM proteases prevented the HIL-6-induced increase in sIL-6R. The inhibition of shedding of mIL-6R had a similar amplitude regardless of which inhibitor was added during the HIL-6 pulse phase or throughout the culture. These results suggest that an ADAM protease may cleave mIL-6R during the early phase of stimulation with HIL-6.6 G; s+ e1 |+ Z

Figure 9. HIL-6 induces shedding of membrane-bound interleukin-6 receptor (mIL-6R) by a disintegrin and metalloprotease (ADAM) proteases in CD133  cells in a phosphatidylinositol-3'-kinase (PI3K)-dependent pathway. 5-FU-pretreated CD133  cells (1.5 x 105 cells per well) were cultured for 2 hours in Stemspan SFT alone, with 100 ng/ml HIL-6, or a combination of HIL-6 and 200 µM TAPI (A). After this period of induction with HIL-6, cells were cultured for 24 hours in HIL-6-free medium supplemented or not with 200 µM TAPI. Supernatants were collected and used for determination of sIL-6R concentration by enzyme-linked immunosorbent assay (ELISA). Results are mean ¡À the standard error of the mean (n = 5¨C12). ***, p

Finally, several studies in the literature suggest the involvement of PI3K and MAPK pathways in the activation of ADAM proteases by phosphorylated JAK. After 5-FU pretreatment, cells were loaded for 2 hours with 50 µM LY294002 and 10 µM U0126 inhibitors then submitted to a 2-hour pulse of HIL-6 (Fig. 9B). These inhibitors did not affect the basal production of sIL-6R. The inhibitor LY294002 partially but significantly (p

HIL-6 Increases LTC-IC Frequency by a Mechanism Involving the Shedding of sIL-6R
$ s+ @* {. ~" J( |  k% c  }
Functional evidence of an autocrine or paracrine loop induced by HIL-6 has resulted from long-term culture of CD133  cells. Cells were cocultured with a stromal cell line in serum-free Stemspan SFT medium. HIL-6 and/or TAPI were added at the beginning of the culture and were not further added, even for the weekly half-medium changes. After 5 weeks of culture, cells were replated in semisolid medium for CFU assay. Limiting dilution analysis of the cultures revealed that LTC-IC frequency was greater after HIL-6 treatment (Table 1). In contrast, TAPI did not significantly modify survival or proliferation of primitive HSCs. In culture with HIL-6, LTC-IC frequency returned to the control value when TAPI was added to the culture.
4 G+ k! i7 {4 ~2 Q% b* ^2 \( [7 ?
Table 1. Limiting dilution analysis of long-term culture of CD133  cells treated with HIL-6 and TAPI+ G2 |' b- l/ R( Y' ^3 v

DISCUSSION
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Several strategies have been developed to isolate the ultimate HSC. Models of candidate stem cells are often based on an antigenic profile with specific antigens, like CD34 or CD133, and/or in the absence of lineage-specific markers. However, phenotypic characterization of HSCs leads to the isolation of a heterogeneous population, which not only contains immature HSCs, but also progenitors and committed cells. Some authors developed alternative models to characterize primitive subsets of HSCs according to their intrinsic properties, like staining with rhodamine-123 and Hoechst 33342 . Cell-cycle analyses showed that, after 5-FU pretreatment in the presence of IL-3, part of the G0 CD133  population was recruited and entered apoptosis.9 T+ q$ T* p3 R- F
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Numerous studies have been performed to determine the expression of mIL-6R on the surface of HSCs, but results are contentious and diverge greatly from 20%¨C90% according the HSC model used by the authors .

In the present study, we demonstrated that pulses with HIL-6 for 2 or 24 hours activated mIL-6R gene expression in CD133  cells. This upregulation of mIL-6R by HIL-6 was also observed at the protein level in untreated or 5-FU-pretreated cells. However, HIL-6 inhibited sIL-6R gene expression on 5-FU-pretreated HSCs after a 2-hour stimulation, while it activated this gene in an unselected population of CD133  cells. This inhibition, specific for a primitive HSC subpopulation, does not impair the presence of sIL-6R in the extracellular media. Moreover, the sIL-6R concentration was greater in supernatants obtained from 5-FU-pretreated CD133  cell cultures after stimulation for 2 hours with HIL-6. This phenomenon does not imply RNA splicing of the IL-6R gene but rather proteolysis of mIL-6R. IL-6R is most likely cleaved by an ADAM protease, ADAM-17, also called TACE (tumor necrosis factor  converting enzyme) and/or ADAM-10 . More interestingly, we have shown that HIL-6 induced the activation of ADAM proteases exclusively in 5-FU-treated CD133  cells. So, primitive HSCs upregulate the mIL-6R gene in response to a transient HIL-6-induced stimulation, but a number of the mIL-6R transported to the membranes are cleaved and released into the extracellular medium.

HIL-6-induced shedding of IL-6R might explain why the expression of mIL-6R did not increase at the surface of the 5-FU-treated HSCs after 24 hours of stimulation with HIL-6, whereas it was observed for the whole CD133  population. Batard et al. have demonstrated that neutralization of endogenous TGF-ß1 by a monoclonal antibody leads to the upregulation of mIL-6R . This effect was not observed in our experimental model, but anti-TGFß1 permitted mIL-6R upregulation by HIL-6 when HIL-6 alone failed to increase mIL-6R expression.

We have identified a subpopulation of proliferating cells derived from an initial CD133  population expressing mIL-6R at a higher rate. Cell-cycle analysis demonstrated that the enhancing effect of HIL-6 on the mIL-6R expression rate was not related to HSC proliferation but to a real upregulation of the IL-6R gene.! a% `& s4 @7 h) ~, @
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The pathways involved in the activation of ADAM proteases were also studied herein. gp130 triggers activation of several intracellular pathways, particularly the JAK/STAT, MAPK, and PI3K pathways , this mechanism is mediated in part by the Ras/MAPK pathway. Nevertheless, we demonstrated that U0126 did not significantly affect shedding of mIL-6R. In regard to this group of arguments, we expect that a PKC isoenzyme may be involved in HIL-6-induced activation of ADAM proteases in HSCs via the PI3K pathway.
1 N( ^# s$ F0 V  M* w4 A- X( n
Surprisingly, the effects of the other gp130 protein¨Crelated cytokines tested were marginal. The production of sIL-6R was not modified when CD133  cells were stimulated by IL-11 or LIF. Expression of mIL-6 transcripts increased slightly, but significantly, in response to LIF stimulation. The differences observed in the effects exerted by the cytokines belonging to the IL-6 family have been previously reported in HSCs and have been attributed to the privileged activation of the MAPK and JAK/STAT pathways by IL-6 compared with IL-11 and LIF .

Long-term culture of HSCs suggests that HIL-6 stimulation triggers autocrine and paracrine loops on HSCs and stromal cells (Fig. 10), and that shedding of mIL-6R takes an important place in this phenomenon. The stimulation of gp130 by HIL-6 induces sIL-6R production by primitive HSCs. The released sIL-6R should bind IL-6, which is constitutively produced by stromal cells and forms complexes that activate the gp130 present on both stromal cells and HSCs. Götze et al. have shown that fused IL-6¨CsIL-6R complex stimulates stroma for survival of HSCs . This factor could be sIL-6R in an autocrine or paracrine loop. This enhanced production of IL-6 is required to balance the increasing number of mIL-6R  HSCs after HIL-6 stimulation. The IL-6¨CsIL-6R complexes can also activate HSCs that do not express mIL-6R by a trans-signaling process. As activation of gp130 is necessary to increase maintenance of HSCs, we assume that HIL-6-induced sIL-6R production is involved in an autocrine loop integrating stromal cells. Cell-cycle analysis suggested that the effect of HIL-6 on the maintenance of primitive HSCs could be attributed to an increase in survival, rather than a promotion of HSC expansion.
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Figure 10. Proposed model for paracrine and autocrine interactions between stromal cells and HSCs in the minimal system of the IL-6 network. The activation of gp130 by IL-6¨CsIL-6R or IL-6¨CmIL-6R complexes leads to the phosphorylation of JAK-2 that activates STAT-1 and STAT-3. The multimeric complex IL-6¨CIL-6R¨Cgp130 is rapidly internalized , and the new gp130  mIL-6R  cells can now respond directly to an IL-6 stimulation. sIL-6R resulting from ADAM protease activation stimulates cells lacking mIL-6R by trans-signaling process. Abbreviations: ADAM, a disintegrin and metalloprotease; gp130, glycoprotein 130; HSC, hematopoietic stem cell; IL-6, interleukin 6; JAK, Janus kinase; mIL-6R, membrane-bound interleukin-6 receptor; PI3K, phosphatidylinositol-3'-kinase; PKC, protein kinase C; sIL-6R, soluble interleukin-6 receptor; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription.

A continuous incubation with HIL-6 failed to increase the level of mIL-6R transcripts. Mechanisms of inhibition may be activated after prolonged stimulation of the gp130 signaling pathway. Numerous inhibitors of the JAK/STAT pathway, such as the cytokine-inducible SH2-containing protein (CIS) and suppressor of cytokine signaling (SOCS)-1, SOCS-2, and SOCS-3, are known to be rapidly induced by IL-6 . This STAT-mediated upregulation of gp130 suggests a mechanism whereby activation of gp130 by its cognate ligands may serve to replenish internalized receptors by de novo protein synthesis. The HIL-6-induced positive feedback may be time controlled by numerous mechanisms of retroinhibition that could render HSCs transiently insensitive to gp130 activation.7 [9 o  `; p: N* i# }

CONCLUSION

We conclude that gp130 activation transiently promotes expression of the mIL-6R gene on HSCs and specifically enhances sIL-6R secretion by proteolytic cleavage of mIL-6R on 5-FU-pretreated CD133  cells. sIL-6R could further activate gp130 on neighboring cells through a trans-signaling process. Subsequently, these cells acquiring the IL-6R  phenotype should be more sensitive to IL-6 and may also commit into differentiation.

ACKNOWLEDGMENTS5 T4 h- I1 i8 _* Q1 t5 ?
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We thank Professor L. Cazin for reviewing the paper, E. Legrand for technical advice on RT-PCR experiments, and Dr. B. Lenormand for giving access to the flow cytometer. D.C. is a recipient of a fellowship from the Minist¨¨re de l¡¯Education Nationale, de l¡¯Enseignement Sup¨¦rieur et de la Recherche of France. This work was partially supported by grants from Association Vie et Espoir and Mission Interminist¨¦rielle Interr¨¦gionale d¡¯Am¨¦nagement du Territoire pour le Bassin Parisien.0 `$ z/ M8 _  D& r% ?
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DISCLOSURES7 g6 F1 J! C0 a

The authors indicate no potential conflicts of interest.
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