Báo cáo Y học: Engineering and use of 32P-labeled human recombinant interleukin-11 for receptor binding studies docx

The binding of 32P-labeled FPDIL-11 to Ba/F3 cells stably transfected with plasmids encoding human IL-11 receptors a and b chains IL-11Ra and gp130 was speci®c and saturable with a high

Engineering and use of 32P-labeled human recombinant interleukin-11 for receptor binding studies

Xiao-Ming Wang1, Jean-Marc Wilkin1, Olivier Boisteau2, Dimitri Harmegnies1, Chrystel Blanc3,

Paul Vandenbussche1, FeÂlix A Montero-Julian3, Yannick Jacques2and Jean Content1

1 Institut Pasteur de Bruxelles, Belgium; 2 Groupe Recherche Cytokines/ReÂcepteurs, UniteÂ, Institut de Biologie, Nantes, France;

3 Immunotech, A Beckman-Coulter Company, Marseille, France

Human interleukin-11 (hIL-11) is a pleiotropic cytokine

that is involved in numerous biological activities such as

hematopoiesis, osteoclastogenesis, neurogenesis and female

fertility IL-11 is obviously a key reagent to study the IL-11

receptors However, conventional radio-iodination

techni-ques lead to a loss of IL-11 bioactivity Here, we report the

construction and the production of a new recombinant

human IL-11 (FPDIL-11) In this molecule, a speci®c

phosphorylation site (RRASVA) has been introduced at

the N-terminus of rhIL-11 It can be speci®cally

phos-phorylated by bovine heart protein kinase and accordingly,

easily radiolabeled with 32P A high radiological speci®c

activity (250 000 c.p.m.
ng)1of protein) was obtained with the retention of full biological activity of the protein The binding of 32P-labeled FPDIL-11 to Ba/F3 cells stably transfected with plasmids encoding human IL-11 receptors

a and b chains (IL-11Ra and gp130) was speci®c and saturable with a high anity as determined from Scatchard plot analysis Availability of this new ligand should prompt further studies on IL-11R structure, expression and regu-lation

Keywords: interleukin-11; cytokines; phosphorylation; radiolabeling; receptor binding

Interleukin-11 (IL-11) is a pleiotropic cytokine that has been

shown to exhibit multiple effects on hematopoietic and

nonhematopoietic systems, including the liver,

gastrointest-inal tract, lung, heart, central nervous system, bone, joint,

and immune system [1] IL-11 has hematopoietic and

thrombocytopoietic activities: in vivo IL-11 administration

stimulates megakaryocyte maturation and increases

periph-eral platelet counts [2] as well as accelerating recovery from

chemotherapy-induced or bone-marrow

transplantation-induced thrombocytopenia [2±6] Numerous experiments

on animal models and clinical trials in patients suffering

from acute and chronic in¯ammatory diseases, including

rheumatoid arthritis [7±9], in¯ammatory liver disease [10],

in¯ammatory bowel disease [11±13], mucositis [14], and

psoriasis [15], have revealed that IL-11 is also an

anti-in¯ammatory and mucosal protective agent Another

important role of IL-11 played in female fertility has been

evidenced by the fact that female mice lacking IL-11

receptor are infertile due to a failure of trophoblast

implantation [16]

IL-11 belongs to the gp130 family of cytokines that includes interleukin-6 (IL-6), viral IL-6 (vIL-6), ciliary neurotropic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin-1 (CT-1), and novel neurotrophin-1/B cell-stimulating factor-3 (NNT-1/ BSF-3) [17±20] These cytokines use the common receptor subunit gp130 for signal transduction by which Janus kinases (Jaks) and transcription factors of the STAT family are activated [21] IL-6 uses a homodimer of gp130 transducer, whereas CNTF, LIF, OSM, CT-1 and NNT-1/BSF-3 assemble a heterodimer of gp130 and another protein LIFR OSM can also recruit a heterodimer of gp130 and OSMR [22] Both LIF and OSM can directly induce heterodimerization of gp130 and LIFR or OSMR, whereas IL-11, IL-6, CNTF and CT-1 must ®rst bind to their speci®c, nonsignaling receptor (named a chain) before inducing dimerization of signal-transducing receptor sub-units (named b chain) In contrast with IL-6, vIL-6 can directly activate gp130 in the absence of IL-6R [23,24] The stoichiometry of the ligand±receptor complex is still unclear for IL-11 Actually, two models have been described One is

a tetrameric complex model suggested by GroÈtzinger et al [25] in which one molecule of IL-11 binds to its speci®c a-receptor via site I of the cytokine and at low concentration

of IL-11±IL-11R complexes, IL-11 recruits two molecules of gp130 through its sites II and III At high IL-11/IL-11R concentrations, it is proposed that the tetramer is able to bind an additional IL-11±IL-11R complex, forming a hexameric, but nonsignaling complex [26] According to

in vitro studies based on immunoprecipitation using differen-tially tagged forms of ligand and soluble receptor compo-nents, a hexameric complex has also been proposed in which two molecules of IL-11, two molecules of IL-11R, either one molecule of gp130 and another still unidenti®ed gp130-type component are involved [27], or two molecules of gp130 as

Correspondence to J Content, Institut Pasteur de Bruxelles, rue

Engeland 642, B-1180, Brussels, Belgium Fax: + 32 2373 32 91,

Tel.: + 32 2373 34 16, E-mail: jcontent@pasteur.be or X.-M Wang,

Fax: + 32 2373 32 91, Tel.: + 32 2373 32 28,

E-mail: xmwang@pasteur.be

Abbreviations: IL, interleukin; IL-11R, interleukin-11 receptor; vIL-6,

viral IL-6; CNTF, ciliary neurotropic factor; LIF, leukemia inhibitory

factor; OSM, oncostatin M; CT-1, cardiotrophin-1;

NNT-1/BSF-3, novel neurotrophin-1/B cell-stimulating factor-3;

DMEM, Dulbecco's modi®ed Eagle's medium; Jaks, Janus kinase.

(Received 19 July 2001, revised 22 October 2001, accepted 22 October

2001)

recently proposed by Barton et al [28] Which one is the

active signalling receptor complex? This question will not be

answered until much more information is available on the

situation within intact cells

The effects of IL-11 must be mediated by the IL-11Ra

and the latter provides ligand speci®city in a functional

multimeric signal transduction complex with gp130 Two

isoforms of the human IL-11R a-chain have been

identi-®ed and cloned [29] They share identical extracellular

and transmembrane domains but differ in their C-terminus

One isoform has a cytoplasmic domain, whereas the second

lacks the entire cytoplasmic domain Both these isoforms

[30] and the soluble IL-11Ra, lacking both the

transmem-brane and cytoplasmic domains [31], were shown to have

similar functional properties, suggesting the dispensability

of these two domains for signaling Structurally, the

extracellular region of the IL-11Ra could be divided into

three domains: an Ig-like domain (D1) and two

®bronectin-type III-like (FNIII) domains (D2 and D3) Recently, most

of the amino-acid residues in IL-11Ra involved in ligand

binding were identi®ed in D3 [32]

In order to provide a convenient IL-11 reagent for the

study of IL-11 cell surface receptors, we describe in this

paper the construction and the production of a new hIL-11

molecule FPDIL-11 and its use to study cell receptor binding

as well as other potential applications provided by this new

reagent

M A T E R I A L S A N D M E T H O D S

Bacterial strains, enzymes and chemicals

Escherichia coli DH5a was from Life BioTechnologies

BL21(DE3) and pET-22b(+) were from Novagen The

catalytic subunit of cAMP-indepentent protein kinase from

bovine heart muscle was obtained from Sigma Human

E coli recombinant IL-11 was from PeproTech Inc

(London, UK) Mouse monoclonal anti-(human gp130) Ig

(B-R3) was from Diaclone Research (BesancËon, France)

MAB628 and polyclonal anti-(hIL-11) Ig (BAF218) were

from R&D Systems [c-32P]ATP, with a speci®c

radio-activity of  3000 Ci
mmol)1, was obtained from

Amer-sham; acrylamide and N,N¢-methylenebisacrylamide were

from Bio-Rad; SDS and monoclonal anti-Flag Ig (M2) were

from Sigma RPMI-1640, Dulbecco's modi®ed Eagle's

medium (DMEM), glutamine, and fetal bovine serum were

from Gibco-BRL

Construction of expression plasmids for recombinant

human IL-11

EcoRI and NotI sites were ®rst introduced by PCR at two

ends of the hIL-11 gene using two primers G310

(5¢-ATCCGGAATTCCCTGGGCCACCACCTGGCCC

CCCT-3¢) and G311 (5¢-ATAGTTTAGCGGCCGCT

TACAGCCGAGTCTTCAGC-3¢) and pIL-11/1 as

tem-plate plasmid To generate the EcoRI±NotI CPDIL11

fragment, which contains an N-terminal Cys (C) and a

bovine heart kinase phosphorylation site (P) as well as a

modi®ed IL-11 lacking the ®rst 10 amino acids (DIL11),

another PCR was performed using two oligonucleotides

YIL11TAG (5¢-ATCCGGAATTCGGTTGTGGTCGT

CGTGCATCTGTTGCATCCCCAG-3¢) and YIL11/Not

I(5¢-ATAGTTTAGCGGCCGCTTACAGCCGAGTCTT CAG-3¢) This fragment was inserted in the vector YepFlag-1 (Kodak Scienti®cImagingSystem)justnextto theFlagtagat the restriction site EcoRI to generate plasmid YepFlag-CPDIL11 The fragment NdeI±NotI (Flag-CPDIL-11) was obtained by the third PCR using two primers G353 (5¢-GGAATTCCATATGGACTACAAGGATGACGATG ACAAG-3¢) and G354 (5¢-ATAGTTTAGCGGCCGCT CACAGCCGAGTCTTCAG-3¢) and the above plasmid as template The expression plasmid pET-FCPDIL11 was constructed by insertion in phase of the fragment NdeI± NotI into the vector pET-22b(+) (Novagen) at the sites NdeI and NotI Because the recombinant protein FCPDIL-11 forms a dimer via the residue Cys and loses the binding activity on cells, another plasmid pET-FPDIL11 was created

by a PCR using two oligonucleotides G390 pGGTCGTCGTGCATCTGTTGC-3¢) and G391 (5¢-pCTTGTCATCGTCATCCTTGTAG-3¢) as primers and the template plasmid pET-FCPDIL11 In this last construct, the Cys residue and the EcoRI site have been deleted All constructs were con®rmed by DNA sequencing

Production and puri®cation of the human recombinant IL-11

The plasmid pET-FPDIL11 was transformed into BL21(DE3) cells E coli cells were cultured in Luria± Bertani medium containing 100 lg
mL)1of ampicillin at

37 °C When the absorbance of growing cells at 600 nm reached  0.6±0.8, the expression of the recombinant protein was induced by addition of 1 mMisopropyl thio-b-D-galactoside for 2 h E coli cells were then harvested and lyzed by sonication for 5 min at an intensity of level 5 using a microprobe (Vibra Cell, Sonics Materials Inc Danburg, Connecticut, USA) in the presence of 0.1% Triton X-100 and 150 lg
mL)1 of lysozyme in 50 mM Hepes, pH 7.4 buffer After two centrifugation cycles at 13 000 g for

25 min at 4 °C, the supernatant was precipitated with (NH4)2SO4at a concentration of 60% saturation in order to concentrate crude proteins Salts were eliminated by dialysis against 50 mMHepes, pH 7.4 buffer before the puri®cation

of samples by chromatography Two puri®cation protocols were used In the ®rst one, a small amount of pure FPDIL-11 was obtained after puri®cation on a Mono-S HR5/5 column (Amersham Pharmacia Biotech) using a 50 mM Hepes buffer, pH 7.4, and a 0±1M NaCl gradient This pure protein was used for labeling and binding assays Another protocol combining chromatography on an SP-Sepharose column using a 50 mMHepes buffer, pH 7.4, and a 0±1M NaCl gradient, and af®nity chromatography on an anti-Flag Ig column allowed the puri®cation of larger amounts of FPDIL-11 We used this preparation to maintain the transfected IL-11-dependent Ba/F3 cells and 7TD1 cells It was also used as competitor in cell receptor binding studies SDS/PAGE and Western blot

SDS/PAGE with 15% polyacrylamide gels was carried out

as described previously [33] After the transfer of proteins from gels onto nitrocellulose ®lters FPDIL-11 was detected

by incubation both with polyclonal anti-(hIL-11) Ig BAF218 and with monoclonal anti-Flag Ig (M2), and ®nally revealed with the alkaline phosphatase system (Sigma)

Cell culture

B13Ra1 and B13Ra2 cells are a murine pro-B lymphocyte

line Ba/F3 stably transfected with plasmids containing two

genes that encode the hIL-11 receptor a and b chains [30]

Cells were maintained in a culture medium RPMI-1640

supplemented with 10% fetal bovine serum, 1% glutamine,

0.8 mg
mL)1G418 (Sigma), 5 lg
mL)1puromycin (Sigma)

and 5 ng
mL)1human IL-11 at 37 °C and 5% CO2 Murine

7TD1 hybridoma cells were cultivated as described

previously [34,35] THP-1 monocytic leukaemia cells

(provided by M Kalai, Ghent University-VIB, Belgium),

K562 chronic myelogenous leukaemia cells (provided by

H Verschueren, Pasteur Institute of Brussels, Belgium), and

CESS myelomonocytic leukaemia cells were maintained in

RPMI-1640 (with glutamine) containing 10% (v/v) fetal

bovine serum MG-63 osteosarcoma cells, A375 metastatic

melanoma cells, HeLa epithelial carcinoma cells, RD

rhabdomyosarcoma cells, and SK-N-MC neuroblastoma

cells (provided by H Verschueren) were maintained in

DMEM containing 10% fetal bovine serum and 2 mM

glutamine All cell lines were maintained at 37 °C and 5%

CO2

IL-11 bioassay

IL-11 activity was measured using the IL-11-dependent

mouse hybridoma cell line 7TD1 These cells were cultivated

in ¯at-bottom microtiter plate that contained 2 ´ 103cells

per well in the presence of twofold dilutions of IL-11

(2 lg
mL)1) After 7 days of culture, the number of

surviving cells was determined by a colorimetric assay of

hexosaminidase In this assay, the absorbance is

propor-tional to the number of cells present in each culture [35]

Each sample was tested in triplicate

Labeling of FPDIL-11 with [c-32P]ATP

FPDIL-11 was labeled through protein phosphorylation

with [c-32P]ATP in the presence of bovine heart kinase

Brie¯y, 1 lg of puri®ed FPDIL-11 was incubated at 30 °C

for 1 h with 0.5 mCi of [c-32P]ATP ( 3000 Ci
mmol)1,

Amersham Corp.) and 6 U of the catalytic subunit of

protein kinase from bovine heart (Sigma) in 80 lL of

20 mM Tris/HCl, pH 7.5, 100 mM NaCl, 12 mM MgCl2,

and 1 mM dithiothreitol The reaction was stopped by

adding 420 lL of 1 mg
mL)1 BSA in a buffer (PPE)

containing 10 mM sodium phosphate, 10 mM sodium

pyrophosphate, and 10 mM EDTA, pH 7.0 at 4 °C The

32P-labeled FPDIL-11 was dialyzed against 3 L of PPE

overnight at 4 °C and then against 1 L of NaCl/Pibuffer for

4 h Incorporation of radioactivity into FPDIL-11 was

measured with a liquid scintillation spectrometer after

precipitation of the protein with 10% trichloroacetic acid

SDS/PAGE of [32P]FPDIL-11 was performed on a slab gel

by the method of Laemmli [33] The purity of [32

P]FPDIL-11 was checked after drying and exposing the gel to an

X-ray ®lm (Kodak XAR) for autoradiography

Binding of [32P]FPDIL-11 to cells

Cells (1 ´ 106) were preincubated in culture medium

lack-ing growth factor for 2 h and were washed three times

with NaCl/Pi, pH 7.4 For binding studies, radiolabeled FPDIL-11 at the indicated concentration in NaCl/Pi containing 0.5% BSA was added to cells The mixture was incubated at 4 °C for the appropriate time and bound radiolabeled FPDIL-11 was separated from the free radio-activity by centrifugation at 3000 g for 1 min through a

0.2-mL layer of a mixture of 40% dioctyl phthalate and 60% dibutyl phthalate (Janssen Chimica, Beerse, Belgium) After quick freezing, the tip of each tube containing the cell pellet was cut-off and radioactivity was counted with a Beckman b-counter Nonspeci®c binding was determined by incubat-ing cells with radiolabeled FPDIL-11 in the presence of a 200-fold molar excess of unlabeled FPDIL-11 The number

of receptors on cells and dissociation constant (Kd) were determined with Scatchard plot analysis according to speci®c binding data

R E S U L T S A N D D I S C U S S I O N Construction and puri®cation of recombinant human IL-11 (FPDIL-11)

Radiolabeled hIL-11 is a useful and very sensitive reagent to study the hIL-11 receptors Human IL-11 labeling with125I has been reported [36], but our numerous attempts to iodinate hIL-11 were unsuccessful due to a loss of bioactivity after labeling As it had been shown that the incorporation of a phosphorylation site into several proteins, such as IFN-a and diphtheria toxin, resulted in a high speci®c radioactivity after 32P-labeling and had no signi®cant effect on their biological activity [37±39], we therefore decided to adopt a similar strategy for IL-11 This strategy is illustrated in Fig 1 The N-terminal nucleotides encoding the ®rst 10 amino acids of IL-11 were deleted and replaced by a sequence encoding a Flag tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) followed by a consensus amino-acid sequence (Arg-Arg-Ala-Ser-Val-Ala) that can be recognized and phosphorylated on the serine residue by the bovine heart kinase [37] The Flag tag was introduced at the end of the molecule to facilitate its puri®cation by af®nity chromatography and immunological detection [40±42] The ®rst 10 amino acids of hIL-11 were deleted in order to keep the size of the recombinant FPDIL-11 similar

to that of hIL-11 and to avoid the problem of expression that may arise in E coli because of the presence of many consecutive proline residues at the N-terminus This deletion was made possible because the ®rst 13 N-terminal amino acids are not necessary for its biological activity and not part

of the sites, as identi®ed by molecular modelling and site-directed mutagenesis, involved in receptor binding [18,43±45]

Fig 1 Nucleotide and amino acid sequences of the N-terminus of human IL-11 and FPDIL-11 The Flag tag is boxed The ®rst 10 amino acids of hIL-11 are bold The phosphorylation site recognized by the bovine heart protein kinase catalytic subunit created in FPDIL-11 is under-lined.

The puri®cation of FPDIL-11 from bacteria consisted of

three main steps: (a) extraction of the recombinant protein,

(b) cation-exchange chromatography, and (c) af®nity

chromatography with anti-Flag Ig coupled to Sepharose

beads One liter of bacterial cell culture yielded 1±2 mg of

puri®ed FPDIL-11 The protein was stable during

puri®ca-tion and no degradapuri®ca-tion was observed after several months

storage at )20 °C The FPDIL-11 protein was pure as

assessed by SDS/PAGE analysis (Fig 2) and Western blots

using antibodies against hIL-11 and Flag peptide (data not

shown) Its apparent molecular mass was  25 kDa, a value

somewhat higher than its real molecular mass (20 kDa)

determined by mass spectroscopy (data not shown) This

difference could be due to the presence within the Flag tag

and the phosphorylation site of numerous charged residues

(1Glu, 5Asp, 2Arg and 2Lys) (Fig 1)

Biological activity of FPDIL-11

The availability of the puri®ed recombinant IL-11 protein

enabled us to test its biological activities in vitro The murine

hybridoma cell line 7TD1 formed by fusion of the mouse

myeloma cell line Sp2/0-Ag14 with spleen cells from a

C57BL/6 mouse was used for this purpose This cell line is

known to respond to picogram amounts of IL-6 [35], but

has also a proliferating response to nanogram amounts of

IL-11 [31] As shown in Fig 3, the recombinant FPDIL-11

had a biological activity very similar to that of wild-type

hIL-11 with an IC50of  0.8 ng
mL)1, con®rming that the

®rst 10 amino acids are dispensable for the biological

activity of IL-11 and indicating that the presence of the Flag

tag as well as the phosphorylation site at the N-terninus

have no detectable effect on the IL-11 functionality

Labeling of FPDIL-11 FPDIL-11 was labeled with [c-32P]ATP using bovine heart protein kinase Autoradiography of the labeled ligand con®rmed the success of the phosphorylation Speci®c radioactivity attained about 250 000 cpm
ng)1of protein, which corresponds to a nearly complete radiophosphate labeling of the IL-11 molecules Speci®city of the labeling was demonstrated using both E coli bacteriophage k protein phosphatase and wild-type hIL-11 Bacteriophage

k phosphatase can hydrolyze phosphate groups on serine, threonineortyrosine-histidineresidues.Incubating[32 P]inter-leukin with this enzyme resulted in the complete loss of its radioactive label (Fig 4A) Human IL-11, which does not contain any putative phosphorylation site, could not be radiolabeled under similar conditions (data not shown) Previous observations have shown that the apparent molecular masses of phosphorylated and nonphosphory-lated proteins are slightly different on SDS/PAGE [46] FPDIL-11 that was phosphorylated with cold ATP in the same conditions as with [c-32P]ATP showed an apparent molecular mass slightly higher than its nonphosphorylated counterpart (Fig 4B), con®rming once more its complete phosphorylation [31P]FPDIL-11 had a biological activity similar to that of wild-type hIL-11, indicating that phosphorylation does not affect the IL-11 functional activity (Fig 3)

Binding of [32P]-FPDIL-11 to cells B13Ra1 and B13Ra2 cells were used to test FPDIL-11 binding to human IL-11 receptors B13Ra1 and B13Ra2 are Ba/F3 cells stably transfected with human gp130 and,

Fig 2 SDS/PAGE of FPDIL-11 Lane 1, 60 lg of the extract

pro-teins from transformed BL21(D3) cells induced by 1 m M isopropyl

thio-b- D -galactoside for 2 h; lane 2, 2 lg of puri®ed FPDIL-11

Pro-teins were stained with Coomassie blue.

Fig 3 Proliferation assay of interleukin-11 on 7TD1 cells IL-11 activity was measured using the mouse hybridoma cell line 7TD1 Brie¯y, 7TD1 cells were cultivated in ¯at-bottom microwell plates containing 2 ´ 10 3 cells per well in the presence of serial dilutions of the cytokine After 7 days of culture, the number of surviving cells was determined by a colorimetric assay of hexosaminidase In this assay, the absorbance is proportional to the number of cells present in each well Each sample was tested in triplicate.

respectively, full length hIL-11Ra and hIL-11Ra lacking the

cytoplasmic domain [30] All binding experiments were

carried out at 4 °C to prevent cell internalization of the

ligand

No speci®c binding of [32P]FPDIL-11 could be detected

on parental Ba/F3 cells The kinetics of the association of

radiolabeled FPDIL-11 with B13Ra1 cells revealed that the

radioligand reached its maximum association to cells after

1-h incubation at 4 °C In subsequent equilibrium binding

studies, [32P]FPDIL-11 was therefore incubated with cells

for 90 min

The dose±response curve of [32P]FPDIL-11 binding to B13Ra1 cells is shown in Fig 5 Nonspeci®c binding component, determined by adding a 200-fold molar excess

of unlabeled FPDIL-11, was low (less than 5% of the total association) Analysis of the speci®c binding data by the method of Scatchard indicated the existence of a single class

of binding sites B13Ra1 cells have  10 550 receptors per cell with an apparent dissociation constant (Kd) of 0.372 nM (Fig 5, inset), which is consistent with that described previously for other cell lines (Kdˆ 300±800 pM) [36,47± 49] Similar results were obtained for B13Ra2 cells (data not shown) We could only detect high af®nity receptors on these cells This suggests either that in these cells gp130 is in excess or that the af®nity of the a subunit is too low to be detected The IL-11 receptor a chain is a transmembrane protein, but its membrane-spanning and cytoplasmic domains are unnecessary for IL-11-induced signal transduc-tion [30,31] As expected, the receptor binding on B13Ra2 cells revealed that the cytoplasmic domain is also dispen-sable for ligand binding

Competition experiments between radiolabeled and unlabeled FPDIL-11 gave an experimental Kiof 0.377 nM, which is similar to the calculated Kdvalue obtained from Scatchard analysis (Fig 6) IL-6 was used as a negative control as this cytokine and IL-11 do not compete for the same receptors [47] When wild-type hIL-11 and [31P]FPDIL-11 were used as competitors, similar results were obtained, suggesting that addition of the Flag tag and phosphorylation site, and phosphorylation of this site at the serine residue as well as deletion of the ®rst 10 amino acids, have no effect on IL-11 binding to the human IL-11Ra and gp130 receptor complex

When 7TD1 cells were used for receptor binding assay, Scatchard analysis of the data revealed that these cells have

 550 receptors per cell with a Kdaround 0.97 nM Human IL-11Ra can interact with murine gp130 and this provides a speci®c high-af®nity binding site for hIL-11 [49] The binding of [32P]FPDIL-11±7TD1 cells expressing both murine IL-11Ra and murine gp130 demonstrated that the interaction between hIL-11 and murine IL-11Ra and murine gp130 was also of high af®nity Taken together, these observations suggest that human and murine IL-11

Fig 5 Binding of radiolabeled ligand to B13Ra1 cells The binding of

[ 32 P]FPDIL-11 to B13Ra1 cells was performed as described under

Materials and methods Speci®c binding (.) represents the di€erence

between total binding (r) and nonspeci®c binding (m) Nonspeci®c

binding represents the binding in the presence of excess unlabeled

FPDIL-11 Values were the means of triplicates from two independent

experiments Standard errors of the means were less than 5% Inset:

Scatchard analysis of FPDIL-11 binding according to speci®c binding

data (bound molecules of 11 were plotted vs bound

FPDIL-11/free FPDIL-11) B max ˆ 10 550 ‹ 200 sites per cell; K d ˆ 0.372 ‹

0021 n M

Fig 4 Phosphorylation of FPDIL-11 (A) [ 32 P]FPDIL-11 (0.8 ng) with 2500 Ci
mmol )1 was treated with or without 5 U of E coli lambda phosphatase for 75 min at 30 °C were separated on SDS/PAGE (15%) After electrophoresis, the gel was dried onto a sheet of Whatman 3 paper and it was then exposed for 10 min to a Kodak X-Omat ®lm (Kodak company) for autoradiography (B) Lane 1, 100 ng of FPDIL-11 phos-phorylated with cold 31 P; lane 2, 1 lg of unphosphorylated FPDIL-11 were separated on a SDS/PAGE (15%) Proteins were coloured by the silver staining method.

receptors are interchangeable for cytokine binding Indeed,

human IL-11Ra shares 82% identity with its murine

homolog [29,36] Although both cytokines display relatively

poor homology in the D1 domain, domains D2 and D3 are

well conserved The Ig-like domain (D1) is not required for

ligand binding as the presence of IL-11 and IL-11R-D2,3 is

suf®cient to induce biological activity [50] The residues

responsible for the ligand binding are mainly located in the

D3, and D2 plays only a minor role [32]

Several human hematopoietic and nonhematopoietic

cell lines (THP-1 monocytic leukaemia cells, K562 chronic

myelogenous leukaemia cells, CESS myelomonocytic

leukaemia cells, MG-63 osteosarcoma cells, A375

meta-static melanoma cells, HeLa epithelial carcinoma cells,

RD rhabdomyosarcoma cells, and SK-N-MC

neuroblas-toma cells) have been tested using [32P]FPDIL-11 to

obtain information about the expression of human IL-11

receptors Speci®c binding was observed in THP-1 and

MG-63 cells and Scatchard plot analysis revealed that

they have,  600 and 800 receptors, respectively, per cell

(data not shown) This is the ®rst time that cell surface

expression of human IL-11 receptors is shown directly in

human cells by use of a radioligand We found the

presence of IL-11Ra on THP-1 cells This is consistent

with previous observations that IL-11 is involved in the

regulation of production of pro-in¯ammatory cytokines

such as TNF-a, IL-1b, IFN-c and IL-12 by monocytes

[55,56], and that the IL-11 receptor analysis on human cell

lines by ¯ow cytometry using monoclonal anti-IL-11Ra Ig

has also revealed the expression of IL-11Ra on these cells

[54] In contrast with previous reports describing IL-11Ra

mRNA detection in K562 cells and skeletal muscle [29],

we did not observe the expression of IL-11Ra on these

cells nor on RD cells MG-63 cells have been shown to

have both IL-11Ra mRNA and the expression of this

receptor [29,54,57] Detection of IL-11R on these cells, in

this study, is in accordance with the fact that IL-11 is able

to induce the formation of osteoclasts in bone morrow

and able to stimulate bone resorption [58]

Inhibition of ligand binding by monoclonal antibodies Monoclonal (IL-11) Ig, gp130 Ig and anti-(IL-11R) Ig were used for antibody competition experi-ments to test whether they would affect FPDIL-11 binding H2 and H56 are two neutralizing anti-(IL-11) Ig that recognize an epitope localized at site II of the cytokine, being the contact point with gp130 These two mAbs were shown

to have an inhibitory effect on IL-11-induced proliferation

of B13Ra1 with an IC50around 3 nMfor H2 and 5 nMfor H56 (C Blanc, I Tacken, J.-M Wilkin, P Vuzio, G Muller-Newen, P.C Heinrich, Y Jacques, J GroÈtzinger, J Content

& F.A Montero-Julian, unpublished results) Similarly, they can also inhibit FPDIL-11 receptor binding with an

IC50of 0.34 nMfor H2 and 0.58 nMfor H56 (Fig 6A) It should be noted that H2 and H56 are not competitive inhibitors of cytokine binding but rather interfere with the formation of the IL-11±IL-11R±gp130 complex The IC50 values of both antibodies that inhibit IL-11-induced proliferation of B13Ra1 cells (3 nMfor H2 and 5 nM for H56) were 10-fold higher than the antibodies concentrations that inhibit [32P]FPDIL-11-receptor binding on the same cells (0.34 nMfor H2 and 0.58 nMfor H56) These results are nevertheless not contradictory because IC50values are not intrinsic constants as they depend on the concentration of ligand used; the higher concentration of ligand used, the larger concentration of inhibitor to compete for 50% of the activity will be needed

B-R3 and MAB628 are two anti-(human gp130) mAbs that interfere with the biological effects of all known cytokines using gp130 as transducing element [30,51±53] Figure 6B shows that B-R3 and MAB628 mAbs inhibit the radioligand binding with IC50 values of 0.47 nM and 0.20 nM, respectively Several mAbs against the human interleukin-11receptora-chainhaverecentlybeenraised[54] I7.4, D14.7, B24.3, D16.1, E24.2, C8.7, and A3.4 recognize the domain III (D3) of IL-11R [54] None of these mAbs are inhibitory of IL-11-induced proliferative response These mAbswere tested inthe FPDIL-11-receptor bindingassay In

Fig 6 Binding of [ 32 P]FPDIL-11 to B13Ra1 cells competed with di€erent interleukin-11 (A) and inhibited by antihuman IL-11 and antihuman gp130 neutralizing antibodies (B) Cells were incubated with the indicated concentrations of, in panel A, unlabeled rhIL-11 (m), unlabeled FPDIL-11 (j), cold labeled [ 31 P]FPDIL-11 (s), and IL-6 (r) as a negative control; in panel B, antihuman IL-11 monoclonal antibodies H2 (.) and H56 (e) and antihuman gp130 monoclonal antibodies MAB628 (h) and B-R3 (n) Data points represent the means of triplicate determinations expressed as a percentage of maximum speci®c binding K i was calculated to be about 0.252 n M for rhIL-11, 0.377 n M for FPDIL-11, and 0.337 n M for [ 31 P]FPDIL-11 These values were obtained by the method of Cheng & Pruso€ [59] IC 50 was calculated to be  0.34 n M for H2, 0.58 n M for H56, 0.20 n M for MAB628, and 0.47 n M for B-R3.

agreement with the proliferation data, none of them had any

inhibitoryeffectonFPDIL-11binding(datanotshown), thus

reinforcing the conclusion that the epitopes recognized by all

these antibodies are distinct from the ligand binding site

The introduction of a phosphorylation site into IL-11 and

other proteins provides a convenient and simple method to

label the proteins to high speci®c radioactivities If multiple

phosphorylation sites were introduced into proteins, much

higher speci®c radioactivities could be generated and

accordingly, this would render the radioligand-detection

much more easier This is a quite useful tool especially in the

case where the expression of certain receptors on the cell

surface is lower The enzymatic labeling by phosphorylation

is a relatively gentle way to radiolabel ligands as compared to

chemical methods that can destroy the biological activity to

some extent Because of its high radioactivity and biological

activity,32P-labeled IL-11 should be a useful reagent for

the characterization and assay of the IL-11 receptors,

pharmacokinetics, diagnostic imaging, and many other uses

A C K N O W L E D G E M E N T S

This work was supported by grants from the European Community

(BIO4 CT 972010), the Fund for Medical Scienti®c Research (contract

3.4611.97, Belgium), the ÔFondation Rose et Jean HoguetÕ and ÔLes amis

de lÕInstitut Pasteur de BruxellesÕ.

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