FEBS 18471
`
`FEBS Letters 407 (1997) 141-147
`
`Reconstitution of two isoforms of the human interleukin-11 receptor and
`comparison of their functional properties
`
`Benoît Lebeaua, Felix A. Montero Julianb, John Wijdenesc, Gerhard Müller-Newend,
`Heike Dahmend, Michel Chérela, Peter C. Heinrichd, Hervé Braillyb, Marie-Martine Halleta,
`Anne Godarda, Stéphane Minviellea, Yannick Jacquesa'*
`aGroupe de Recherche Cytokines et Récepteurs, Unité INSERM 463, 9 Quai Moncousu, 44035 Nantes Cedex 1, France
`hImmunotech SA, 130 Avenue De Lattre de Tassigny, 13276 Marseille Cedex 9, France
`cDiaclone, 1 Boulevard Fleming, 25020 Besançon Cedex, France
`A Institut für Biochemie, RWTH, Pauwelsstrasse 30, 52057 Aachen, Germany
`
`Received 3 February 1997; revised version received 17 March 1997
`
`Abstract Long-term stable Ba/F3 transfectants (B13Ral and
`B13Roc2) expressing two
`isoforms of the human
`I L - l l Ra
`receptor ( al full length or a2 lacking the cytoplasmic domain)
`in combination with human gpl30 were established. I L - l l R od
`and I L - l l R a2 were each expressed and detected as three bands
`upon Western blot analysis, with apparent molecular masses in
`agreement with those of the polypeptide backbone (47 and 44
`kDa, respectively) with no, one or two N-linked sugars. B13Rocl
`and B13Ra2 bound IL-11-thioredoxin with similar efficiencies
`and proliferated with superimposable dose-response curves to IL-
`11, demonstrating that the intracellular domain of I L - l l Ra has
`no significant contribution on ligand binding and signaling.
`Analysis of a set of anti-human gpl30 mAbs confirmed the
`similar responsiveness of B13Rocl and B13Rct2 transfectants.
`© 1997 Federation of European Biochemical Societies.
`
`Key words: H u m an interleukin-11 receptor; Isoform;
`Transfection (Ba/F3 cells); Proliferation; Monoclonal
`antibody
`
`1. Introduction
`
`Interleukin-11 (IL-11) is a cytokine produced by a variety
`of cells from mesenchymal origin such as
`fibroblasts, kerati-
`nocytes, chondrocytes, synoviocytes, bone marrow stromal
`cells and osteoblasts. It also displays multiple biological activ-
`ities, many of which are similar to those exerted by interleu-
`kin-6 (IL-6) and, to a lesser extent, by other pleiotropic cyto-
`kines
`such as L IF
`(leukemia
`inhibitory
`factor), O SM
`(oncostatin M) and C N TF (ciliary neurotrophic factor) [ 1-
`4]. F or example, IL-11 and IL-6 act in synergy with IL-3 to
`support the growth of pluripotent bone marrow progenitors
`and to stimulate megakaryogenesis [5]. They both regulate the
`synthesis and secretion of acute-phase proteins by hepatocytes
`[6], promote the differentiation of B lymphocytes into immu-
`noglobulin-secreting cells [7], function as adipogenesis inhib-
`itory factors [8] and participate in the development of osteo-
`clastic
`cells
`[9]. The molecular
`identification
`of
`the
`components of the functional high-affinity receptors for these
`pleiotropic cytokines has provided some explanation for this
`redundancy of action. All cytokines of this family require the
`
`"Corresponding author. Fax: (33) 2-40-35-66-97
`
`Abbreviations: IL-llRa, interleukin-11 receptor; Trx, thioredoxin;
`s-IL-6Ra, soluble IL-6 receptor; mAb, monoclonal antibody
`
`transduce
`to
`transmembrane glycoprotein
`common gpl30
`their biological activities. The gpl90 glycoprotein (low-affinity
`LIF receptor) is also required for signal transduction through
`L I F, O SM (type I), C N TF and CT-1 receptors [10,11].
`In the case of the C N T F, IL-6 and IL-11 receptors, addi-
`tional receptor components (a chains) have been described
`which confer cytokine specificity. The C N T F - Ra chain is a
`72 k Da glycoprotein (gp72) which is attached to the plasma
`membrane through a glycophosphatidyl inositol (GPI) anchor
`[12]. The IL-6Rcc chain is a 80 k Da membrane anchored gly-
`coprotein with a 82 amino acids intracellular domain [13].
`More recently IL-11 Ret chains have been described in mouse
`[14] and human [15,16]. We have shown the existence of two
`human I L - l l Ra c D N As resulting from alternative exon splic-
`ing [15,17]. The first c D NA encodes a transmembrane glyco-
`protein containing, like the IL-6Rcc chain, a cytoplasmic do-
`main (32 amino acids). The second c D NA isoform encodes a
`protein lacking, like the C N T F - Ra chain, a cytoplasmic do-
`main. In this study, we describe the establishment and char-
`acterization of stable Ba/F3 cell lines transfected with the two
`human I L - l l Ra isoforms and use these cells to evaluate the
`contribution of the intracytoplasmic domain of I L - l l Ra
`in
`ligand binding and signal transduction.
`
`2. Experimental procedures
`
`2.1. Expression constructs
`Human IL-11 cDNA was subcloned into pTrxFus (Invitrogen BV,
`Leek, The Netherlands) to construct a thioredoxin-IL-11
`fusion
`cDNA. Expression was performed in E. coli and the fusion protein
`(IL-11-Trx) was prepared as a crude extract from E. coli osmotic
`shock treatment [18]. EcoR\
`fragment of BQM15.C cDNA clone
`containing the human IL-11 Reel coding sequence was inserted into
`the pLXSPuro (Transgen) eukaryotic expression vector to obtain
`pLXSP/IL-HRal and selected via the puromycin-resistance gene. Hu-
`man IL-llRa2 complete coding sequence was obtained by PCR am-
`plification with the oligonucleotides, 5'-GCGGAATTCATGAGG-
`GACCAATGGCAGT and 5'-GGACTTTCTAGATGCTGGCAC,
`using BQP32.21 cDNA clone as template and ligated to the EcoR.1-
`Xba\ fragment of BQM15.C cDNA. The sequence of the PCR frag-
`ment was confirmed by double-strand DNA sequencing. This insert
`was subcloned in pLXSPuro to obtain pLXSP/IL-HRcc2.
`
`2.2. DNA transfection experiments
`and Ba/F3/gpl30/IL-llRa2
`Ba/F3/gpl30/IL-llRal
`(B13Ral)
`(B13Ra2) were established by electroporating (at 900 uF and 300
`V) mouse pro-B-cell Ba/F3 with 20 ug of pLXSP/IL-HRocl or
`pLXSP/IL-HRa2 and 20 ug of pRCNeo/. Transfected cells were se-
`lected in a culture medium containing: RPMI-1640, 10% fetal calf
`serum, 1% glutamine, 0.8 mg/ml G418 (Sigma-Aldrich, St. Quentin
`
`0014-5793/97/S17.00 © 1997 Federation of European Biochemical Societies. All rights reserved.
`P / / S 0 0 1 4 - 5 7 9 3 ( 9 7 ) 0 0 3 2 6 -8
`
`Lassen - Exhibit 1067, p. 1
`
`

`

`142
`
`B. Lebeau et al.lFEBS Letters 407 (1997) 141-147
`
`Fallavier, France), 5 (ig/ml puromycin (Sigma) and 5 ng/ml (0.25 nM)
`human IL-11. Ba/F3/IL-llRal (BRal), Ba/F3/IL-llRa2 (Bra2) and
`Ba/F3/gpl30 (B13) were obtained using the same procedure except
`that cells were only transfected with one construct and selected in
`medium containing 10% of WEHI-3-conditioned medium as a source
`of IL-3 instead of IL-11 and either 5 ug/ml puromycin (BRal, BRa2)
`or 0.8 mg/ml G418 (B13).
`
`labeled goat anti-mouse antiserum (1:200; Immunotech, Marseille,
`France). For I L - l l Ra staining, cells were incubated with IL-11-Trx
`(30 nM), washed 3 times, further incubated for 1 h with a mouse anti-
`Trx antibody (1:5000) (Invitrogen), washed again (3 times), and fi-
`nally stained with a phycoerythrin-linked goat anti-mouse antiserum
`(Immunotech). In some experiments, a 42-fold molar excess of human
`IL-11 (1.25 uM) was added together with IL-11-Trx.
`
`2.3. Cytokines and monoclonal antibodies
`Human E. coli recombinant IL-11 was from Peprotech Inc. (Lon-
`don, UK). IL-6, sIL-6Ra and the anti-IL-6Ra monoclonal antibody
`(mAb) PM1 were kindly given by Dr. Yasukawa (Tosoh Corp., To-
`kyo, Japan). The anti-human gpl30 mAbs B-P8, B-S12, B-R3, B-P4
`and the anti-human IL-6Rrx mAb B-N12 were from Diaclone (Besan-
`çon, France). Their initial characterisation and analysis of functional
`properties have already been described [19-21].
`
`2.4. Proliferation assays
`Transfected Ba/F3 cells were washed twice in culture medium lack-
`ing growth factor and seeded in 96-well microtiter plates at a density
`of 15 X 103 cells in 50 JJ.1 per well. In a first type of assay, cells were
`incubated (in a final volume of 100 |il) with serial dilutions either of
`IL-11 (5 nM), of IL-6 (20 nM) in the presence or not of sIL-6Ra
`(fixed concentration of 8 nM), or of a mixture of B-P8 (6.66 nM) plus
`B-S12 (20 nM). In a second type of assay, cells were stimulated with
`IL-11 (0.25 nM) or IL-6 (0.6 nM) plus sIL-6Ra (8 nM), and serial
`dilutions of B-N12 (20 nM), PM-1 (20 nM), B-R3 (20 nM) or B-P4
`(20 nM). Assays were performed in triplicate. After incubation at
`37°C for 36 h, cellular proliferation was assessed by a (4,5-dimethyl-
`thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) based assay
`(Sigma).
`
`2.5. Flow cytometric analysis
`Cells (1 X105) were preincubated for 3 h in culture medium lacking
`growth factor and washed 3 times. For gpl30 staining, the B-R3 mAb
`was added at a final concentration of 67 nM and incubated on ice for
`1 h. After washing (3 times), cells were stained with a phycoerythrin-
`
`2.6. Immunoblot analysis
`Cells were washed 3 times in 10 ml of phosphate-buffered saline
`(PBS) and pelleted by centrifugation. Pellets were frozen (—80°C) and
`thawed once, homogenized in a Dounce homogenizer, and centrifuged
`for 20 min at 2500 rpm at 4°C. The supernatant was centrifuged for
`30 min at 14000 rpm at 4°C. Pellets were resuspended in 10 mM Tris
`buffer (pH 7.4), containing leupeptin at 0.1 mg/ml (Sigma). The pro-
`tein concentration was determined by the Bicinchoninic Acid (BCA)
`method (Pierce). Membrane proteins (50 u.g/lane) were resolved by
`10% SDS-PAGE and transferred onto a PVDF membrane (Amer-
`sham, Arlington Heights, USA). After blocking, the membrane was
`incubated with a rabbit polyclonal antibody (1:100) directed against a
`20-amino-acid peptide of murine IL-11R (Santa Cruz Biotechnology,
`Santa Cruz, CA). Detection was performed using chemiluminescence
`(ECL kit, Amersham).
`
`3. Results
`
`long-term
`
`transfectants with
`3.1. Generation ofBalFS
`proliferation
`in response
`to
`IL-11
`The murine Ba/F3 cell line has been widely used to study
`the reconstitution of cytokine receptors [22]. It is absolutely
`dependent on IL-3 for growth and does not proliferate
`in
`response to IL-6 or L I F. H u m an I L - l l R al or
`I L - l l R a2
`c D N As were transfected
`in Ba/F3 cells either alone or
`in
`combination with full-length h u m an g p l 30 c D N A. Trans-
`fected cells were subsequently cultured in selection media con-
`
`Fig. 1. Flow cytometric analysis of human gpl30: B-R3 mAb and phycoerythrin-conjugated second antibody (open area). Control: second anti-
`body alone (closed area).
`
`"*"
`10°
`
`10
`
`Lassen - Exhibit 1067, p. 2
`
`

`

`B. Lebeau et al.lFEBS Letters 407 (1997) 141-147
`
`143
`
`10
`FL2-H
`Fig. 2. Flow cytometric analysis of human IL-llRa: IL-11-Trx/anti-Trx antibody and phycoerythnn-conjugated second antibody in the ab-
`sence (thin lane, open area) or presence (thick lane, open area) of a 42-fold molar excess of human IL-11. Control: anti-Trx plus second anti-
`body (closed area)
`
`taining appropriate antibiotic(s) and IL-3 conditioned me-
`dium or 5 ng/ml of human recombinant IL-11. In the presence
`of IL-3, cellular expansion was observed in all transfected
`cultures. In IL-11 containing medium, cellular expansion
`was only observed from Ba/F3 cells co-transfected with IL-
`l l R al
`and gpl30
`(B13Rod) or
`IL-llRa2 and gpl30
`(B13Roc2). Furthermore, when cells selected in IL-3 condi-
`tioned medium were shifted to IL-11 containing medium,
`only cells co-transfected with gpl30 and IL-llRal or IL-
`11 Ra2 continued to grow, whereas cells transfected without
`insert, with IL-11 Rot 1 alone (BRccl), IL-llRoc2 alone (BRa2)
`or gpl30 alone (B13) rapidly died. These results were consist-
`ently obtained in several independent transfection experi-
`ments. Long-term proliferation of B13Ral and B13Roc2
`were observed. These cell lines are now routinely grown (for
`more than 6 months) in IL-11 containing medium and retain
`their IL-11 growth-dependency (see Fig. 4A).
`
`3.2. Analysis of the expression of the transfected receptor
`chains
`Ba/F3 cells expressing different combinations of the trans-
`fected cDNAs were established as stable cell lines. The level of
`expression at the cell surface of the transfection products was
`evaluated by flow cytometry and Western blots. Fig. 1 shows
`the flow cytometric histograms obtained with an anti-gpl30
`mAb (B-R3). In contrast to the parental BaF3 cell line which
`was negative, B13, B13Ral and B13Rcc2 expressed gpl30
`with similar intensities. Due to the lack of a suitable anti-
`IL-llRcx for flow cytometric analysis, the expression of IL-
`
`l l Ra was measured with the combination of an IL-11-thio-
`redoxin fusion protein (IL-11-Trx) and an anti-Trx antibody.
`As shown in Fig. 2, both B13Rocl and B13Roc2 transfectants
`were similarly labeled with IL-11-Trx, whereas Ba/F3 and
`B13 cells were negative. The specificity of IL-11-Trx binding
`was assessed by the observation that a 42-fold molar excess of
`unlabeled recombinant IL-11 almost completely inhibited this
`binding.
`Western blots were performed on membrane preparations
`from these different cell lines, using a polyclonal antibody
`raised against a N-terminal peptide of murine IL-llRa (Fig.
`3). This antibody was negative on parental Ba/F3 cell lines
`and B13 cells and very clearly identified specific bands on IL-
`HRa/gpl30 transfected cells. Three bands with molecular
`masses of 47, 50 and 54 kDa were detected on cells transfected
`with the IL-llRal isoform, whereas on IL-llRa2 transfec-
`tants, these three bands had lower masses of 44, 47 and 51
`kDa, respectively.
`
`3.3. Proliferative responses
`We next examined the proliferative response of the various
`transfectants to different cytokines. All cell lines proliferated
`in response to IL-3-conditioned medium with comparable
`dose responses (data not shown). Only IL-llRa/gpl30 dou-
`ble-transfected cells did proliferate in response to IL-11 (Fig.
`4A), showing that both IL-llRa and gpl30 are required and
`sufficient for IL-11 signaling in Ba/F3 cells. The dose-response
`curves relative to the effects of IL-11 on the B13Ral and
`B13Ra2 proliferative responses were almost superimposable,
`
`Lassen - Exhibit 1067, p. 3
`
`

`

`144
`
`B. Lebeau et al.lFEBS Letters 407 (1997) 141-147
`
`B-P4 antibody abbrogated the IL-11 response in B13Rod and
`B13Ra2 cell lines with a similar efficacy as B-R3 (ICsos = 0.24
`and 0.18 nM) (Fig. 5G). Unexpectedly, the B-P4 antibody
`also abbrogated the IL-6/sIL-6Roc proliferative effect on the
`two cell lines (IC50s = 0.43 and 0.47 nM) (Fig. 5H).
`
`4. Discussion
`
`This study shows that co-expression in the Ba/F3 cell line of
`the human IL-11 Rot with human gpl30 is necessary and suf-
`ficient for the acquisition of human IL-11 mediated prolifer-
`ative response and for the establishment of Ba/F3 cell trans-
`fectants which can be grown on a long-term basis under the
`growth promoting effect of recombinant human IL-11. These
`transfectants are now grown in our laboratory for more than
`6 months without any significant changes of their IL-11
`growth response sensitivities. These results extend recent ob-
`
`Fig. 3. Western blot analysis of human IL-llRa. Molecular weight
`markers (left) are in kDa.
`
`thereby indicating that the two isoforms have comparable
`efficiencies in complementing gpl30 for signaling. The concen-
`tration of IL-11 inducing half maximal proliferative responses
`(IC50) were 97 and 118 pM, respectively, values compatible
`with a high-affinity interaction.
`The responses of these two double transfectants to the com-
`bination of IL-6 plus sIL-6Roe were also comparable (Fig.
`4B), a result further indicating that the two cell lines express
`equivalent amounts of functional human gpl30. The IC50S for
`IL-6 in the presence of a fixed concentration of sIL-6Roc (8
`nM) were 66 and 75 pM, respectively, values in agreement
`with its high-affinity equilibrium binding constant [23]. We
`also tested two anti-human gpl30 mAbs (B-P8 and B-S12)
`which have been described to display synergistic and agonistic
`functions [19]. In agreement with these findings, B-P8 plus B-
`S12 induced the proliferation of B13Rccl and B13Roc2 trans-
`fectants (Fig. 4C) with efficiencies comparable to those in-
`duced by IL-11 or IL-6/sIL-6Ra. Again, the responses of
`the two transfectants were superimposable, with IC50S of
`0.38 and 0.41 nM.
`IL-11 versus IL-6/sIL-6Ra proliferative responses of the
`two transfectants were further assessed by the use of addi-
`tional monoclonal antibodies (Fig. 5). B-R3 is an anti-human
`gpl30 mAb which interferes with the biological effects of all
`known cytokines using gpl30 as transducing element, whereas
`B-P4 is an anti-gpl30 mAb which has been proposed to in-
`terfere specifically with the IL-11 response [19-21]. PM1 and
`B-N12 are anti-human IL-6Roc mAbs. PM1 is a blocking anti-
`body [24], whereas B-N12 is not (J. Wijdenes, unpublished
`result). For each mAb tested, the responses of B13Rocl and
`B13Ra2 were always almost superimposable. The B-N12 anti-
`body had no effect in either system (Fig. 5A,B). The PM1
`mAb inhibited the IL-6/sIL-6Roc responses (IC 5os: 6.3 and
`7.5 nM) (Fig. 5D) while only slightly diminishing the IL-11
`driven proliferation (Fig. 5C). The proliferation induced by
`either IL-11 or IL-6/sIL-6Roc was totally abbrogated by mAb
`B-R3 (Fig. 5E,F). The IC 50s describing the inhibitory effects
`on IL-11 were 0.30 and 0.24 nM, and those on the IL-6
`response were 0.44 and 0.41 nM. These values are in full
`agreement with the binding affinity of B-R3 on these cells as
`measured by Scatchard analysis (K^ = 0.43 and 0.49 nM on
`B13Rotl and B13Ra2 cells respectively; data not shown). The
`
`0.001
`
`0.01
`
`0.1
`IL-11 (nM)
`[ ■ 1 111 n n j — - T - r T T m r r ™ - - T T T T T T]
`
`1
`
`1 M
`
`IIIUJ
`
`1.4
`
`1.2
`l
`
`I
`
`0.8
`
`o
`in
`Q 0.6
`° 0.4
`0.2
`0
`
`+ SIL-6R0C
`
`(8nM)
`
`0.001
`
`0.01
`
`I
`
`II
`
`
`
`1
`
`l
`
`0.1
`IL-6 (nM)
`
`I
`
`1
`
`I
`
`10
`
`0.01
`
`1
`0.1
`(B-P8 + B-S12)(nM)
`
`10
`
`Fig. 4. Proliferative responses of B13Ral (•) and B13Ra2 (o) cells.
`
`Lassen - Exhibit 1067, p. 4
`
`

`

`B. Lebeau et al.lFEBS Letters 407 (1997) 141-147
`
`145
`
`IL-11
`
`IL-6 + sIL-6Ra
`
`o
`a
`
`a
`o
`r—
`v->
`Q
`O
`
`Q
`O
`
`I o
`
`IT)
`Q
`O
`
`Fig. 5. Effects of anti-gpl30 and anti-IL-6R mAbs on B13Ral (•) and B13Ra2 (o) proliferative responses induced either by IL-11 (5 nM)
`(left) or by IL-6 (0.6 nM) plus sIL-6Ra (8 nM) (right).
`
`1
`0.1
`B-P4 (nM)
`
`1
`0.1
`B-P4 (nM)
`
`servations on similarly co-transfected Ba/F3 cells showing that
`IL-11 was able to maintain cell viability on a short (48 h) time
`period [14,16] and provide for the first time the description of
`stable human IL-1 IRa/gp 130 Ba/F3 cell transfectants prolif-
`erating in response to human IL-11.
`We have previously shown that the human IL-11 Roc chain
`exists in two different
`isoforms resulting from alternative
`
`splicing of a single gene. These two naturally occurring IL-
`U Ra isoforms differ only by the presence of the 32-amino-
`acids cytoplasmic domain [15]. We took advantage of this
`situation to analyze the functional implication of this cyto-
`plasmic domain in the Ba/F3 system.
`A polyclonal antibody raised against the N-terminal peptide
`(residues 25^14) of mouse IL-11 Ret was shown to react with
`
`Lassen - Exhibit 1067, p. 5
`
`

`

`146
`
`B. Lebeau et al.lFEBS Letters 407 (1997) 141-147
`
`human I L - l l Ra in Western blot. This could be expected from
`the high degree of amino-acid sequence homology (95%)
`found in this region between mouse and human receptors
`[15]. This antibody enabled us to document for the first
`time the biochemical features of the human IL-11 Roc chain.
`It reacted with three bands of molecular masses of 47, 50 and
`54 kDa on the B13Ral cells and with three bands of molec-
`ular masses of 44, 47 and 51 kDa on the B13Roc2 cells. This
`pattern is consistent both with the predicted molecular masses
`of the mature proteins encoded by the human I L - l l R al and
`IL-llRa2 cDNA isoforms, which are respectively 43 100 and
`39800 Da, and with the existence within the extracellular
`domain of the human IL-llRa of two potential N-glycosyla-
`tion sites [15]. Thus, for each receptor isoform, the lower band
`likely corresponds to the unglycosylated species, whereas the
`upper bands would correspond to glycosylated species con-
`taining one or two N-linked sugars, respectively. Indeed, the
`increments in molecular mass from the lower to the upper
`band are in the range of 3^1 kDa, values consistent with
`the average molecular mass of 2800 Da calculated for a com-
`plex N-linked carbohydrate [25]. When comparing the two
`receptor isoforms, the difference in molecular masses between
`two paired bands is about 3 kDa, a value compatible with
`that predicted from the difference in amino acids (32 amino
`acids; 3500 Da).
`
`The two B13Rccl and B13Ra2 cell lines expressed similar
`amounts of the human gpl30 signal transducer as revealed by
`flow cytometry. They were also similarly able to bind the
`fusion protein IL-11-Trx. When the proliferative responses
`to human IL-11 were compared, the two transfectants were
`found to display nearly superimposable dose responses with
`high affinity (IC50 around 100 pM). Taken together, these
`data strongly suggest that, at least in the Ba/F3 transfection
`system, both IL-llRa
`isoforms similarly conjugate with
`gpl30 to bind IL-11 and transduce a proliferative signal.
`The intracellular domain of the human IL-llRa chain seems
`therefore dispensable for binding to and signaling through the
`receptor complex in this cellular system.
`In the case of the IL-6 receptor, homodimerisation of the
`gpl30 subunit has been shown to be the key event in signal
`transduction and the IL-6Ra cytoplasmic domain is consid-
`ered to play no role in the signaling process. This concept is
`based on the observations that soluble forms of the extracel-
`lular domain of IL-6Ra can combine with IL-6 and gpl30 to
`trigger the signaling event, and that the intracellular domain
`of IL-6Ra lacks characteristic sequence motifs required for
`signaling [26]. However, analysis of the function of the IL-
`6Ra in hepatoma cells has indicated that, whereas its cyto-
`plasmic domain is not absolutely required for signal trans-
`duction (C-reactive protein promoter activation), the last 40
`amino acids of this domain contribute to maximal IL-6 re-
`sponse [27]. Similarly, soluble form of the mouse IL-llRa
`chain were found to be less effective than the membrane
`form in hepatoma cells, embryonal carcinoma cells and T
`lymphocytes [28], suggesting an auxiliary function for the
`membrane and/or cytoplasmic domains. Our present results
`with the human IL-11 receptor system in Ba/F3 cells clearly
`demonstrate that the intracellular domain has no influence on
`the efficiency of signaling.
`Various anti-gpl30 mAbs have already been described and
`analysed for their ability to interfere with the biological activ-
`ities mediated by cytokines using the gpl30 transducing sub-
`
`unit. Among them are the B-P8 and B-S12 mAbs which have
`been shown to display agonistic and synergistic activities on
`XG1 myeloma and TF1 erythroleukemia cell lines and on
`stem cells [19]. Our data show that this agonistic activity is
`also observed in Ba/F3 cells transfected with gpl30, confirm-
`ing these previous findings. The B-R3 mAb was shown to
`inhibit the growth promoting activity of IL-6, CNTF, LIF
`and OSM on the XG4-CNTF myeloma cell line as well as
`the growth inducing effect of IL-11 on the XG-6-IL-11 mye-
`loma cell line [19,21]. This mAb also inhibited the growth
`promoting effects of all these cytokines, also including CT-1,
`on the TF1 cell line and haptoglobin secretion induced by IL-
`6 and OSM on hepatoma HepG2 cells [20]. In agreement with
`these observations, B-R3 was found in this study to inhibit the
`proliferation of the two IL-llRa transfectants induced either
`by IL-11 or the combination of IL-6 plus sIL-6Ra. The in-
`hibitory capacities of B-R3 on the IL-11 or IL-6 signals were
`similar (IC50 around 0.3 nM and 0.4 nM, respectively) and in
`agreement with its binding constant (K& around 0.45 nM) on
`both cell types. This observation supports the notion that B-
`R3 is not a competitive inhibitor of cytokine binding but
`rather interferes with a region of gpl30 important for its
`dimerisation. B-P4 is another anti-human gpl30 mAb which
`has been described to specifically inhibit (with respect to other
`cytokines) the IL-11 induced proliferative response of myelo-
`ma cell lines [19] and TF1 cells [20]. This antibody was indeed
`found in this study to be as efficient as B-R3 in inhibiting the
`IL-11 responses of B13Ral and B13Ra2 transfectants (IC5o
`around 0.2 nM). However, it also inhibited the proliferation
`of these transfectants in response to IL-6 plus sIL-6Ra with
`an efficiency again comparable to that of B-R3 (IC50 around
`0.45 nM). As a control, the anti-IL6Ra mAb PM1 only af-
`fected the IL-6 response. These results therefore suggest that
`on transfected Ba/F3 cells, the epitope defined by B-P4 might
`be used in common by the IL-11/IL-llRa and IL-6/IL-6Ra
`complexes to activate signal transduction. The discrepancy of
`our findings with previous observations [19-21] might be
`linked to the fact that we used a soluble form of IL-6Ra,
`whereas the other reports were dealing with membrane-anch-
`ored IL-6Ra. It could also reflect differences in sensitivity
`among various IL-6-induced biological responses to the block-
`ing effect of B-P4. This discrepancy might also indicate that
`the conformations of the reconstituted receptors in Ba/F3 cells
`differ in some way with those of natural receptors constitu-
`tively expressed by cell lines. However it may be, elucidation
`of this apparent discrepancy deserves further investigations.
`
`Acknowledgements: This work was supported by the Institut National
`de la Santé et de la Recherche Médicale (INSERM), the Centre Na-
`tional de la Recherche Scientifique (CNRS) and the Association pour
`la Recherche contre le Cancer (ARC) (grant #6474). B.L. is a recip-
`ient of a fellowship from the Association pour la Recherche en Im-
`munologie et Cancérologie (ARIC, Nantes). The authors thank Mrs
`Isabelle Corre (INSERM U463 Nantes) for expert technical assistance
`and Dr. Jean François Moreau (Bordeaux) for helpful discussion.
`
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