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Trophic effects of cardiotrophin-1 and interleukin-11 on rat dorsal root ganglion neurons in vitro
Molecular Brain Research 64 Ž1999. 80–84
Research report
Trophic effects of cardiotrophin-1 and interleukin-11 on rat dorsal root ganglion neurons in vitro Michael Thier a , Mark Hall b, John K. Heath b, Diane Pennica c , Joachim Weis
d,)
a
d
Institute of Neuropathology, Technical UniÕersity (RWTH), Aachen, Germany b School of Biochemistry, UniÕersity of Birmingham, Edgbaston, UK c Department of Molecular Oncology, Genentech, South San Francisco, CA, USA DiÕision of Neuropathology, Institute of Pathology, UniÕersity of Bern, Murtenstr. 31, 3010 Bern, Switzerland Accepted 24 November 1998
Abstract Cardiotrophin-1 ŽCT-1. was originally isolated for its hypertrophy inducing effects on cardiac myocytes whereas interleukin-11 ŽIL-11. was identified due to its ability to stimulate an interleukin-6 ŽIL-6. dependent plasmocytoma cell line. Both cytokines are structurally and functionally related to a group of factors called neuropoietic cytokines, which also includes IL-6, ciliary neurotrophic factor ŽCNTF., leukemia inhibitory factor ŽLIF., and oncostatin M. These factors have trophic effects on subsets of neurons. In the present study we examined the influence of CT-1 and IL-11 on newborn rat dorsal root ganglion neuron survival in vitro. Mouse CT-1 showed prominent trophic effects that were comparable to those of CNTF and LIF. Mouse IL-11 alone did not enhance neuronal survival, but soluble mouse IL-11 receptor a rendered neurons sensitive to IL-11. Surprisingly, soluble IL-11 receptor a even had slight neurotrophic effects by itself. These results suggest that CT-1 and IL-11 might also be involved in the physiological regulation of sensory neuron survival. Thus, they might, like CNTF, become tools for the therapeutic intervention in neurodegeneration due to disease, toxicity, and trauma. q 1999 Elsevier Science B.V. All rights reserved. Keywords: CT-1; IL-11; Soluble receptor; Neurotrophic; Neuropoietic cytokine; Sensory neuron
1. Introduction Cardiotrophin-1 ŽCT-1. and interleukin-11 ŽIL-11. are members of the ‘neuropoietic’ cytokine family which also includes ciliary neurotrophic factor ŽCNTF., leukemia inhibitory factor ŽLIF., oncostatin M, and interleukin-6 ŽIL6.. These molecules have overlapping bioactivities and share a common helical framework w19x. Intracellular signalling by these cytokines either requires homodimerization of their common signal transducing receptor subunit gp130 or heterodimerization of gp130 with a further b-receptor component such as LIF receptor ŽLIFR.. The receptor complexes for IL-6, CNTF, IL-11, and CT-1 each contain a third, ligand-specific primary a-receptor subunit ŽIL-6R, CNTFR, IL-11R, and CT-1R, respectively.. These can be released from the cell surface and confer sensitivity
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for the ligands to cells that themselves do not express the a-receptor components w19x. CT-1 was initially identified and cloned because of its ability to induce cardiac myocyte hypertrophy w13,14x. CT-1 acts on ciliary, sympathetic, and motor neurons similar to CNTF w14,15x and is expressed in embryonic mouse brain, spinal cord, dorsal root ganglia ŽDRG., skeletal muscle, and skin w13,17x, indicating that CT-1 might be a target derived andror autocrinerparacrine trophic factor for motor and sensory neurons. However, it is unknown so far if CT-1 is trophic for sensory DRG neurons. IL-11 was first isolated as a bone marrow fibroblast-derived cytokine that stimulates proliferation of IL-6-dependent cell lines w21x. IL-11 stimulates the neuronal differentiation of embryonic hippocampal cell lines w8x. The expression pattern of IL-11 in brain and muscle suggests that cells in these tissues are targets for IL-11 w5,12x. However, in contrast to CNTF and LIF, IL-11 had only minimal effects on the phenotype of sympathetic neurons and failed to promote DRG neuron survival in vitro w4,18x.
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 3 2 9 - 5
M. Thier et al.r Molecular Brain Research 64 (1999) 80–84
The neurotrophic effects of CNTF are better characterized than those of CT-1 and IL-11. CNTF promotes the survival of sensory, autonomic, hippocampal, striatal, cerebellar, and motor neurons, making CNTF a candidate molecule for the treatment of neurodegenerative disorders w16x. However, CNTF lacks a conventional signal peptide and cannot be secreted by mammalian cells w16x. In contrast, CT-1 and IL-11 are secreted by cells and might therefore be more relevant for the physiological regulation of neuronal survival than CNTF w15,21x. Here we used newborn rat DRGs, for which neuronal survival during development, after axotomy, and in culture largely depends on the availability of neurotrophic proteins such as nerve growth factor ŽNGF., brain derived neurotrophic factor ŽBDNF., or CNTF and LIF w3,9,10,18x. We show that mouse CT-1 is as effective as human CNTF in promoting the survival of cultured newborn rat DRG neurons. Mouse IL-11 had a minor, but significant effect on rat DRG neurons when soluble Žs. mouse IL-11Ra was also present. Moreover, sIL-11Ra exerted significant neurotrophic activity by itself.
2. Materials and methods Recombinant mouse CT-1 was produced in 293 cells and purified as described previously w13x. Recombinant human CNTF was produced in Escherichia coli as described w20x. E. coli-derived recombinant mouse IL-11 was commercially obtained from R & D Systems ŽBad Nauheim, Germany.. E. coli-derived recombinant human NGF was purchased from Boehringer Mannheim ŽMannheim, Germany.. Two forms of recombinant soluble mouse IL-11Ra were expressed in 293tsA1609neo cells and purified as described w6x, one dimeric form consisting of the ectodomain of mouse IL-11Ra linked to human Fc ŽsIL11R-Fc. and one monomeric form ŽsIL-11R. that was cleaved from the Fc portion of sIL-11Ra-Fc using an HRV protease. Newborn rat DRG neurons were cultured as described w20x with minor modifications. Ganglia were incubated for 45 min at 378C in Ca2q- and Mg 2q-free phosphate buffered saline containing papain Ž9 unitsrml; Sigma, St. Louis, MO, USA., DNase I Ž200 unitsrml; Boehringer Mannheim., D,L-cysteine-HCl Ž0.2 mgrml; Sigma., bovine serum albumin Ž0.2 mgrml; Boehringer Mannheim., and glucose Ž5 mgrml; Gibco, Gaithersburg, MD, USA.. After enzymatic and mechanical dissociation, the single cell suspensions were enriched for neurons by preplating for 2–3 h and by two-fold centrifugation at 900 rpm for 5 min. Approximately 400 cells were seeded into each well of polyornithinerlaminin-coated 96-well flat bottom tissue culture plates into serum-free Dulbecco’s modified Eagle medium ŽGibco; a31966. containing the recombinant proteins. After 50 h in culture, cells were stained with the
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vital dye MTT ŽSigma. for 45 min. Staining was blocked by adding 10 ml 35% paraformaldehyde and viable neurons were counted using an inverted microscope. According to morphological criteria Žsize, presence of neurites., approximately 95% of surviving cells were neurons. Experiments were performed in at least triplicate wells and repeated for three or more times. The t-test for paired samples was used for statistical analysis.
3. Results CNTF and mouse CT-1 enhanced the survival of the cultured DRG neurons in a dose-dependent fashion ŽFig. 1.. At CT-1 concentrations equal to or above 0.1 ngrml the number of surviving neurons was significantly different from control values Ž t-test, p - 0.01.. The maximal number of surviving neurons was equal to the numbers obtained with human CNTF. When the concentrations yielding half maximal survival were compared, mouse CT-1 was approximately equipotent to human CNTF in promoting rat DRG neuron survival. Human NGF at 10 ngrml was even more potent than CNTF and CT-1, respectively ŽFig. 1.. Approximately 10% of the initially seeded neurons survived without exogenous trophic proteins. Mouse IL-11 alone failed to promote the survival of newborn rat DRG neurons at physiologically relevant concentrations ŽFig. 2A.. Similarly, the dimeric soluble mouse IL-11 a-receptor ŽsIL-11R-Fc. alone did not show any effects at 1 mgrml Žnot shown.. However, at 1 mgrml this soluble receptor rendered neurons sensitive to IL-11 ŽFig. 2A.; at 100 ngrml mouse IL-11 in the presence of 1 mgrml mouse sIL-11R-Fc, the difference in the number of surviving neurons compared to controls became statisti-
Fig. 1. Concentration-dependent survival promoting activities of CNTF ŽI., CT-1 Ž'., and NGF ŽB., on newborn rat DRG neurons in vitro. Cells were cultured in the presence of increasing amounts of recombinant proteins. The data are means of at least three separate assays in triplicate wells Ž nG9..
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compared to the effects of human CNTF, approximately 250-fold higher concentrations of IL-11 in the presence of 1 mgrml sIL-11R were needed to obtain similar results.
4. Discussion
Fig. 2. ŽA. Neurotrophic effects of CNTF ŽI., IL-11 Ž'., IL-11qsIL11R-Fc ŽB., and sIL-11R-Fc Žl.. Cells were cultured in the presence of different amounts of recombinant proteins. Data of three individual assays in triplicate wells were plotted Ž ns9.. ŽB. Survival promoting activities of CNTF ŽI., IL-11 Ž'., IL-11qsIL-11R ŽB., and sIL-11R Žl.. Cells were cultured in the presence of increasing amounts of recombinant proteins. The data are means of three separate assays in triplicate wells Ž ns9..
cally significant Ž t-test, p - 0.01.. The maximal number of surviving neurons using IL-11 and sIL-11R-Fc did not reach the level that was yielded with half maximal concentrations of human CNTF ŽFig. 2A.. Surprisingly, the monomeric soluble mouse IL-11 a-receptor ŽsIL-11R. at 1 mgrml promoted the survival of DRG neurons by itself ŽFig. 2B.. Compared to controls without recombinant proteins, the survival of neurons was enhanced approximately 1.7-fold. This value was significantly different from control values Ž t-test, p - 0.01.. In combination with increasing amounts of IL-11, the monomer was approximately 9-fold more potent than the dimeric form of soluble IL-11 receptor ŽFig. 2B.. The molecular weight of sIL-11R-Fc is about twice as high as that of sIL-11R w7x. Thus, when molar concentrations of the soluble receptors in combination with IL-11 would be compared, sIL-11R was at least 4-fold more potent than sIL-11R-Fc in yielding similar survival rates. However,
Following peripheral nerve injury and in the course of neurodegenerative diseases, neurons die by a mechanism resembling programmed cell death or apoptosis. The exogenous, therapeutic application of trophic molecules might promote the survival of degenerating neurons and enhance nerve regeneration w7x. Several growth factors including CNTF and LIF have already been shown to prevent the death of neurons and to promote neurite extension subsequent to neuronal injury in vivo and in vitro. Our results demonstrate that mouse CT-1 at concentrations likely to occur in vivo is as effective on newborn rat DRG neurons as human CNTF. After two days, the neuron-enriched cultures contained approximately 95% neuronal cells indicating that the observed cytokine effects were not mediated indirectly by glial cells. Thus, these data suggest that DRG neurons express high affinity receptors for CT-1. CT-1 mRNA expression has been found in mouse skeletal muscle and spinal cord, but not in motor neurons w13,15,17x. CT-1 promotes motor neuron survival in vitro and in vivo and—in contrast to CNTF—can be secreted by mammalian cells w15x. These results indicate that CT-1 might be a target derived andror paracrine neurotrophic factor for rodent motor neurons. Similarity, CT-1 is expressed in the skin and DRGs of mice w17x. We here show that CT-1 also promotes the survival of rat DRG neurons suggesting that CT-1 might play a role as target-derived andror autocrinerparacrine neurotrophic factor for sensory neurons as well. To our knowledge, auto- or paracrine neurotrophic effects of neuropoietic cytokines have not been demonstrated with certainty so far. However, most newborn rat DRG neurons die within two days in culture, but some survive without exogenous trophic support for several days. This ‘basal’ neuronal survival might be due to other auto- or paracrine trophic factors, such as BDNF w1x, but also might be due to low levels of neuropoietic cytokines such as CNTF, LIF, and CT-1. As mentioned above, CNTF, LIF, and CT-1 are potent trophic factors for several neuronal cell types in vitro and in vivo. IL-11, thus far, has been shown to stimulate the neuronal differentiation in immortalized embryonic hippocampal cell lines w8x. Compared to CNTF and LIF, IL-11 has only minimal effects on sympathetic and sensory neurons in vitro w4,18x. In the present study, we have shown that mouse IL-11 can significantly enhance the survival of cultured rat DRG neurons if soluble mouse IL-11R is also present. However, when compared to the effects of human CNTF, approximately 250-fold higher concentrations of IL-11 in the presence of sIL-11R are still needed to obtain similar survival rates. Albeit soluble
M. Thier et al.r Molecular Brain Research 64 (1999) 80–84
mouse IL-11R preparations are active on cells of rat and human origin w2x, we cannot exclude the possibility that species differences prevent a high affinity interaction of the mouse IL-11rsIL-11R complex with the secondary rat receptor molecules. Considering the potent, species-independent actions of human CNTF, rat CNTF, human LIF, mouse LIF, and mouse CT-1 Žpresent results and w3,9,10,18x., this possibility seems to be rather unlikely. IL-11 requires an IL-11-specific a-receptor component and gp130 for signal transduction w2,5,6x. Furthermore, Neddermann et al. w11x recently demonstrated that in human cells human IL-11 is unable to induce either gp130 homodimerization or gp130rLIFR heterodimerization. Therefore, an as yet unidentified a-receptor chain might be involved in human IL-11 signaling. Whether a similar b-subunit is also involved in rodent IL-11 signaling has not been determined so far. However, the lack of an as yet unidentified receptor component might explain the weak trophic effects of mouse IL-11rsIL-11R on newborn rat DRG neurons. In the present study, the monomeric mouse sIL-11R had trophic effects for rat DRG neurons by itself. This finding was somewhat surprising, because signal-inducing activities of primary neuropoietic cytokine a-receptor subunits without the respective cytokines have not yet been demonstrated. A simple explanation for this finding would be that DRG neurons produce and secrete IL-11 by themselves, which might then activate intracellular signalling if soluble IL-11 receptor components are provided by other cells. A similar high basal activity of mouse sIL-11R has previously been observed on human HepG2 hepatoma cells that express and secrete IL-11 w2x. It is important to note that we cannot exclude that human IL-11 expressed innately by 293 cells might have copurified with the mouse sIL-11R and induced slight effects on rat cells without exogenous IL-11. Bioactivity of human IL-11 via mouse sIL-11R on rat cells has already been demonstrated w2x. However, in this case the dimeric soluble mouse IL-11-receptor, sIL11R-Fc, should also exert effects by itself, because both soluble receptor protein preparations used here were produced identically with the exception of the cleavage of the monomeric sIL-11R from the Fc portion w6x. Thus, the trophic activity of sIL-11R alone most probably cannot be ascribed to copurified IL-11 or other impurities in the soluble receptor preparations. Due to its trophic effects on motor neurons in vitro and in vivo, the neuropoietic cytokine CNTF is discussed as a drug to treat human motor neuron disease w7,16x. The observations that CT-1 and IL-11 plus soluble IL-11R also act as neurotrophic factors therefore suggest that the application of CT-1 or IL-11 might be a further therapeutic option to treat motor or sensory neurons affected by neuropathy. Moreover, a combined application of several neuropoietic cytokines or combinations with neurotrophins might be an option to overcome side effects induced by the application of one cytokine at high doses.
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Acknowledgements We thank Dr. D. Suter and Dr. J. Weissenberger for critically reading the manuscript. Support: IZKF CNS of the RWTH Aachen. This study was presented as part of the doctoral thesis of M. Thier ŽRWTH Aachen, 1998..
References w1x A. Acheson, J.C. Conover, J.P. Fandl, T.M. DeChiara, M. Russell, A. Thadani, S.P. Squinto, G.D. Yancopoulos, R.M. Lindsay, A BDNF autocrine loop in adult sensory neurons prevents cell death, Nature 374 Ž1995. 450–453. w2x H. Baumann, Y. Wang, K.K. Morella, C.-F. Lai, H. Dams, D.J. Hilton, R.G. Hawley, A. Mackiewicz, Complex of the soluble IL-11 receptor and IL-11 acts as IL-6-type cytokine in hepatic and nonhepatic cells, J. Immunol. 157 Ž1996. 284–290. w3x S.S. Cheema, L. Richards, M. Murphy, P.F. Bartlett, Leukemia inhibitory factor prevents the death of axotomised sensory neurons in the dorsal root ganglia of the neonatal rat, J. Neurosci. Res. 37 Ž1994. 213–218. w4x M.-J. Fann, P.H. Patterson, Neuropoietic cytokines and activin A differentially regulate the phenotype of cultured sympathetic neurons, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 43–47. w5x D.J. Hilton, A.A. Hilton, A. Raicevic, S. Rakar, M. Harrison-Smith, N.M. Gough, C.G. Begley, D. Metcalf, N.A. Nicola, T.A. Willson, Cloning of a murine IL-11 receptor Ž-chain: requirement of gp130 for high affinity binding and signal transduction, EMBO J. 13 Ž1994. 4765–4775. w6x J. Karow, K.R. Hudson, M.A. Hall, A.B. Vernallis, J.A. Taylor, A. Gossler, J.K. Heath, Mediation of interleukin-11-dependent biological responses by a soluble form of the interleukin-11 receptor, Biochem. J. 318 Ž1996. 489–495. w7x R.M. Lindsay, Therapeutic potential of the neurotrophins and neurotrophin-CNTF combinations in peripheral neuropathies and motor neuron disease, in: Growth Factors as Drugs for Neurological and Sensory Disorders. Ciba Found. Symp., Vol. 196, Wiley, Chichester, 1996, pp. 39–53. w8x M.F. Mehler, R. Rozental, M. Dougherty, D.C. Spray, J.A. Kessler, Cytokine regulation of neuronal differentiation of hippocampal progenitor cells, Nature 362 Ž1993. 62–65. w9x P.K. Mulderry, Neuropeptide expression by newborn and adult rat sensory neurons in culture: effects of nerve growth factor and other neurotrophic factors, Neuroscience 59 Ž1994. 673–688. w10x M. Murphy, K. Reid, M.A. Brown, P.F. Bartlett, Involvement of leukemia inhibitory factor and nerve growth factor in the development of dorsal root ganglion neurons, Development 117 Ž1993. 1173–1182. w11x P. Neddermann, R. Graziani, G. Ciliberto, G. Paonessa, Functional expression of soluble human interleukin-11 ŽIL-11. receptor and stoichiometry of in vitro IL-11 receptor complexes with gp130, J. Biol. Chem. 271 Ž1996. 30986–30991. w12x H. Neuhaus, B. Bettenhausen, P. Bilinski, D. Simon-Chazottes, J.-L. Guenet, A. Gossler, Etl2, a novel putative type-I cytokine receptor ´ expressed during mouse embryogenesis at high levels in skin and cells with skeletogenic potential, Dev. Biol. 166 Ž1994. 531–542. w13x D. Pennica, K.L. King, K.J. Shaw, E. Luis, J. Rullamas, S.-M. Luoh, W.C. Darbonne, D.S. Knutzon, R. Yen, K.R. Chien, J.B. Baker, W.I. Wood, Expression cloning of cardiotrophin 1, a cytokine that induces cardiac myocyte hypertrophy, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 1142–1146. w14x D. Pennica, K.J. Shaw, T.A. Swanson, M.W. Moore, D.L. Shelton, K.A. Zioncheck, A. Rosenthal, T. Taga, N.F. Paoni, W.I. Wood, Cardiotrophin-1, J. Biol. Chem. 270 Ž1995. 10915–10922.
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w15x D. Pennica, V. Arce, T.A. Swanson, R. Vejsada, R.A. Pollock, M. Armanini, K. Dudley, H.S. Phillips, A. Rosenthal, A.C. Kato, C.E. Henderson, Cardiotrophin-1, a cytokine present in embryonic muscle, supports long-term survival of spinal motoneurons, Neuron 17 Ž1996. 63–74. w16x M. Sendtner, P. Carroll, B. Holtmann, R.A. Hughes, H. Thoenen, Ciliary neurotrophic factor, J. Neurobiol. 25 Ž1994. 1436–1453. w17x Z. Sheng, D. Pennica, W.I. Wood, K.R. Chien, Cardiotrophin displays early expression in the murine heart tube and promotes cardiac myocyte survival, Development 122 Ž1996. 419–428. w18x R. Simon, M. Thier, A. Kruttgen, S. Rose-John, O. Weiergraber, ¨ ¨
P.C. Heinrich, J.M. Schroder, J. Weis, Human CNTF and related ¨ cytokines: effects on DRG neurone survival, Neuroreport 7 Ž1995. 153–157. w19x T. Taga, T. Kishimoto, Gp130 and the interleukin-6 family of cytokines, Annu. Rev. Immunol. 15 Ž1997. 797–819. w20x M. Thier, R. Simon, A. Kruttgen, S. Rose-John, P.C. Heinrich, J.M. ¨ Schroder, J. Weis, Site-directed mutagenesis of human CNTF: func¨ tional analysis of recombinant variants, J. Neurosci. Res. 40 Ž1995. 826–835. w21x Y.-C. Yang, Interleukin 11: an overview, Stem Cells 11 Ž1993. 474–486.
Research report
Trophic effects of cardiotrophin-1 and interleukin-11 on rat dorsal root ganglion neurons in vitro Michael Thier a , Mark Hall b, John K. Heath b, Diane Pennica c , Joachim Weis
d,)
a
d
Institute of Neuropathology, Technical UniÕersity (RWTH), Aachen, Germany b School of Biochemistry, UniÕersity of Birmingham, Edgbaston, UK c Department of Molecular Oncology, Genentech, South San Francisco, CA, USA DiÕision of Neuropathology, Institute of Pathology, UniÕersity of Bern, Murtenstr. 31, 3010 Bern, Switzerland Accepted 24 November 1998
Abstract Cardiotrophin-1 ŽCT-1. was originally isolated for its hypertrophy inducing effects on cardiac myocytes whereas interleukin-11 ŽIL-11. was identified due to its ability to stimulate an interleukin-6 ŽIL-6. dependent plasmocytoma cell line. Both cytokines are structurally and functionally related to a group of factors called neuropoietic cytokines, which also includes IL-6, ciliary neurotrophic factor ŽCNTF., leukemia inhibitory factor ŽLIF., and oncostatin M. These factors have trophic effects on subsets of neurons. In the present study we examined the influence of CT-1 and IL-11 on newborn rat dorsal root ganglion neuron survival in vitro. Mouse CT-1 showed prominent trophic effects that were comparable to those of CNTF and LIF. Mouse IL-11 alone did not enhance neuronal survival, but soluble mouse IL-11 receptor a rendered neurons sensitive to IL-11. Surprisingly, soluble IL-11 receptor a even had slight neurotrophic effects by itself. These results suggest that CT-1 and IL-11 might also be involved in the physiological regulation of sensory neuron survival. Thus, they might, like CNTF, become tools for the therapeutic intervention in neurodegeneration due to disease, toxicity, and trauma. q 1999 Elsevier Science B.V. All rights reserved. Keywords: CT-1; IL-11; Soluble receptor; Neurotrophic; Neuropoietic cytokine; Sensory neuron
1. Introduction Cardiotrophin-1 ŽCT-1. and interleukin-11 ŽIL-11. are members of the ‘neuropoietic’ cytokine family which also includes ciliary neurotrophic factor ŽCNTF., leukemia inhibitory factor ŽLIF., oncostatin M, and interleukin-6 ŽIL6.. These molecules have overlapping bioactivities and share a common helical framework w19x. Intracellular signalling by these cytokines either requires homodimerization of their common signal transducing receptor subunit gp130 or heterodimerization of gp130 with a further b-receptor component such as LIF receptor ŽLIFR.. The receptor complexes for IL-6, CNTF, IL-11, and CT-1 each contain a third, ligand-specific primary a-receptor subunit ŽIL-6R, CNTFR, IL-11R, and CT-1R, respectively.. These can be released from the cell surface and confer sensitivity
) Corresponding author. [email protected]
Fax:
q 41-31-632-4995;
E-mail:
for the ligands to cells that themselves do not express the a-receptor components w19x. CT-1 was initially identified and cloned because of its ability to induce cardiac myocyte hypertrophy w13,14x. CT-1 acts on ciliary, sympathetic, and motor neurons similar to CNTF w14,15x and is expressed in embryonic mouse brain, spinal cord, dorsal root ganglia ŽDRG., skeletal muscle, and skin w13,17x, indicating that CT-1 might be a target derived andror autocrinerparacrine trophic factor for motor and sensory neurons. However, it is unknown so far if CT-1 is trophic for sensory DRG neurons. IL-11 was first isolated as a bone marrow fibroblast-derived cytokine that stimulates proliferation of IL-6-dependent cell lines w21x. IL-11 stimulates the neuronal differentiation of embryonic hippocampal cell lines w8x. The expression pattern of IL-11 in brain and muscle suggests that cells in these tissues are targets for IL-11 w5,12x. However, in contrast to CNTF and LIF, IL-11 had only minimal effects on the phenotype of sympathetic neurons and failed to promote DRG neuron survival in vitro w4,18x.
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 3 2 9 - 5
M. Thier et al.r Molecular Brain Research 64 (1999) 80–84
The neurotrophic effects of CNTF are better characterized than those of CT-1 and IL-11. CNTF promotes the survival of sensory, autonomic, hippocampal, striatal, cerebellar, and motor neurons, making CNTF a candidate molecule for the treatment of neurodegenerative disorders w16x. However, CNTF lacks a conventional signal peptide and cannot be secreted by mammalian cells w16x. In contrast, CT-1 and IL-11 are secreted by cells and might therefore be more relevant for the physiological regulation of neuronal survival than CNTF w15,21x. Here we used newborn rat DRGs, for which neuronal survival during development, after axotomy, and in culture largely depends on the availability of neurotrophic proteins such as nerve growth factor ŽNGF., brain derived neurotrophic factor ŽBDNF., or CNTF and LIF w3,9,10,18x. We show that mouse CT-1 is as effective as human CNTF in promoting the survival of cultured newborn rat DRG neurons. Mouse IL-11 had a minor, but significant effect on rat DRG neurons when soluble Žs. mouse IL-11Ra was also present. Moreover, sIL-11Ra exerted significant neurotrophic activity by itself.
2. Materials and methods Recombinant mouse CT-1 was produced in 293 cells and purified as described previously w13x. Recombinant human CNTF was produced in Escherichia coli as described w20x. E. coli-derived recombinant mouse IL-11 was commercially obtained from R & D Systems ŽBad Nauheim, Germany.. E. coli-derived recombinant human NGF was purchased from Boehringer Mannheim ŽMannheim, Germany.. Two forms of recombinant soluble mouse IL-11Ra were expressed in 293tsA1609neo cells and purified as described w6x, one dimeric form consisting of the ectodomain of mouse IL-11Ra linked to human Fc ŽsIL11R-Fc. and one monomeric form ŽsIL-11R. that was cleaved from the Fc portion of sIL-11Ra-Fc using an HRV protease. Newborn rat DRG neurons were cultured as described w20x with minor modifications. Ganglia were incubated for 45 min at 378C in Ca2q- and Mg 2q-free phosphate buffered saline containing papain Ž9 unitsrml; Sigma, St. Louis, MO, USA., DNase I Ž200 unitsrml; Boehringer Mannheim., D,L-cysteine-HCl Ž0.2 mgrml; Sigma., bovine serum albumin Ž0.2 mgrml; Boehringer Mannheim., and glucose Ž5 mgrml; Gibco, Gaithersburg, MD, USA.. After enzymatic and mechanical dissociation, the single cell suspensions were enriched for neurons by preplating for 2–3 h and by two-fold centrifugation at 900 rpm for 5 min. Approximately 400 cells were seeded into each well of polyornithinerlaminin-coated 96-well flat bottom tissue culture plates into serum-free Dulbecco’s modified Eagle medium ŽGibco; a31966. containing the recombinant proteins. After 50 h in culture, cells were stained with the
81
vital dye MTT ŽSigma. for 45 min. Staining was blocked by adding 10 ml 35% paraformaldehyde and viable neurons were counted using an inverted microscope. According to morphological criteria Žsize, presence of neurites., approximately 95% of surviving cells were neurons. Experiments were performed in at least triplicate wells and repeated for three or more times. The t-test for paired samples was used for statistical analysis.
3. Results CNTF and mouse CT-1 enhanced the survival of the cultured DRG neurons in a dose-dependent fashion ŽFig. 1.. At CT-1 concentrations equal to or above 0.1 ngrml the number of surviving neurons was significantly different from control values Ž t-test, p - 0.01.. The maximal number of surviving neurons was equal to the numbers obtained with human CNTF. When the concentrations yielding half maximal survival were compared, mouse CT-1 was approximately equipotent to human CNTF in promoting rat DRG neuron survival. Human NGF at 10 ngrml was even more potent than CNTF and CT-1, respectively ŽFig. 1.. Approximately 10% of the initially seeded neurons survived without exogenous trophic proteins. Mouse IL-11 alone failed to promote the survival of newborn rat DRG neurons at physiologically relevant concentrations ŽFig. 2A.. Similarly, the dimeric soluble mouse IL-11 a-receptor ŽsIL-11R-Fc. alone did not show any effects at 1 mgrml Žnot shown.. However, at 1 mgrml this soluble receptor rendered neurons sensitive to IL-11 ŽFig. 2A.; at 100 ngrml mouse IL-11 in the presence of 1 mgrml mouse sIL-11R-Fc, the difference in the number of surviving neurons compared to controls became statisti-
Fig. 1. Concentration-dependent survival promoting activities of CNTF ŽI., CT-1 Ž'., and NGF ŽB., on newborn rat DRG neurons in vitro. Cells were cultured in the presence of increasing amounts of recombinant proteins. The data are means of at least three separate assays in triplicate wells Ž nG9..
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compared to the effects of human CNTF, approximately 250-fold higher concentrations of IL-11 in the presence of 1 mgrml sIL-11R were needed to obtain similar results.
4. Discussion
Fig. 2. ŽA. Neurotrophic effects of CNTF ŽI., IL-11 Ž'., IL-11qsIL11R-Fc ŽB., and sIL-11R-Fc Žl.. Cells were cultured in the presence of different amounts of recombinant proteins. Data of three individual assays in triplicate wells were plotted Ž ns9.. ŽB. Survival promoting activities of CNTF ŽI., IL-11 Ž'., IL-11qsIL-11R ŽB., and sIL-11R Žl.. Cells were cultured in the presence of increasing amounts of recombinant proteins. The data are means of three separate assays in triplicate wells Ž ns9..
cally significant Ž t-test, p - 0.01.. The maximal number of surviving neurons using IL-11 and sIL-11R-Fc did not reach the level that was yielded with half maximal concentrations of human CNTF ŽFig. 2A.. Surprisingly, the monomeric soluble mouse IL-11 a-receptor ŽsIL-11R. at 1 mgrml promoted the survival of DRG neurons by itself ŽFig. 2B.. Compared to controls without recombinant proteins, the survival of neurons was enhanced approximately 1.7-fold. This value was significantly different from control values Ž t-test, p - 0.01.. In combination with increasing amounts of IL-11, the monomer was approximately 9-fold more potent than the dimeric form of soluble IL-11 receptor ŽFig. 2B.. The molecular weight of sIL-11R-Fc is about twice as high as that of sIL-11R w7x. Thus, when molar concentrations of the soluble receptors in combination with IL-11 would be compared, sIL-11R was at least 4-fold more potent than sIL-11R-Fc in yielding similar survival rates. However,
Following peripheral nerve injury and in the course of neurodegenerative diseases, neurons die by a mechanism resembling programmed cell death or apoptosis. The exogenous, therapeutic application of trophic molecules might promote the survival of degenerating neurons and enhance nerve regeneration w7x. Several growth factors including CNTF and LIF have already been shown to prevent the death of neurons and to promote neurite extension subsequent to neuronal injury in vivo and in vitro. Our results demonstrate that mouse CT-1 at concentrations likely to occur in vivo is as effective on newborn rat DRG neurons as human CNTF. After two days, the neuron-enriched cultures contained approximately 95% neuronal cells indicating that the observed cytokine effects were not mediated indirectly by glial cells. Thus, these data suggest that DRG neurons express high affinity receptors for CT-1. CT-1 mRNA expression has been found in mouse skeletal muscle and spinal cord, but not in motor neurons w13,15,17x. CT-1 promotes motor neuron survival in vitro and in vivo and—in contrast to CNTF—can be secreted by mammalian cells w15x. These results indicate that CT-1 might be a target derived andror paracrine neurotrophic factor for rodent motor neurons. Similarity, CT-1 is expressed in the skin and DRGs of mice w17x. We here show that CT-1 also promotes the survival of rat DRG neurons suggesting that CT-1 might play a role as target-derived andror autocrinerparacrine neurotrophic factor for sensory neurons as well. To our knowledge, auto- or paracrine neurotrophic effects of neuropoietic cytokines have not been demonstrated with certainty so far. However, most newborn rat DRG neurons die within two days in culture, but some survive without exogenous trophic support for several days. This ‘basal’ neuronal survival might be due to other auto- or paracrine trophic factors, such as BDNF w1x, but also might be due to low levels of neuropoietic cytokines such as CNTF, LIF, and CT-1. As mentioned above, CNTF, LIF, and CT-1 are potent trophic factors for several neuronal cell types in vitro and in vivo. IL-11, thus far, has been shown to stimulate the neuronal differentiation in immortalized embryonic hippocampal cell lines w8x. Compared to CNTF and LIF, IL-11 has only minimal effects on sympathetic and sensory neurons in vitro w4,18x. In the present study, we have shown that mouse IL-11 can significantly enhance the survival of cultured rat DRG neurons if soluble mouse IL-11R is also present. However, when compared to the effects of human CNTF, approximately 250-fold higher concentrations of IL-11 in the presence of sIL-11R are still needed to obtain similar survival rates. Albeit soluble
M. Thier et al.r Molecular Brain Research 64 (1999) 80–84
mouse IL-11R preparations are active on cells of rat and human origin w2x, we cannot exclude the possibility that species differences prevent a high affinity interaction of the mouse IL-11rsIL-11R complex with the secondary rat receptor molecules. Considering the potent, species-independent actions of human CNTF, rat CNTF, human LIF, mouse LIF, and mouse CT-1 Žpresent results and w3,9,10,18x., this possibility seems to be rather unlikely. IL-11 requires an IL-11-specific a-receptor component and gp130 for signal transduction w2,5,6x. Furthermore, Neddermann et al. w11x recently demonstrated that in human cells human IL-11 is unable to induce either gp130 homodimerization or gp130rLIFR heterodimerization. Therefore, an as yet unidentified a-receptor chain might be involved in human IL-11 signaling. Whether a similar b-subunit is also involved in rodent IL-11 signaling has not been determined so far. However, the lack of an as yet unidentified receptor component might explain the weak trophic effects of mouse IL-11rsIL-11R on newborn rat DRG neurons. In the present study, the monomeric mouse sIL-11R had trophic effects for rat DRG neurons by itself. This finding was somewhat surprising, because signal-inducing activities of primary neuropoietic cytokine a-receptor subunits without the respective cytokines have not yet been demonstrated. A simple explanation for this finding would be that DRG neurons produce and secrete IL-11 by themselves, which might then activate intracellular signalling if soluble IL-11 receptor components are provided by other cells. A similar high basal activity of mouse sIL-11R has previously been observed on human HepG2 hepatoma cells that express and secrete IL-11 w2x. It is important to note that we cannot exclude that human IL-11 expressed innately by 293 cells might have copurified with the mouse sIL-11R and induced slight effects on rat cells without exogenous IL-11. Bioactivity of human IL-11 via mouse sIL-11R on rat cells has already been demonstrated w2x. However, in this case the dimeric soluble mouse IL-11-receptor, sIL11R-Fc, should also exert effects by itself, because both soluble receptor protein preparations used here were produced identically with the exception of the cleavage of the monomeric sIL-11R from the Fc portion w6x. Thus, the trophic activity of sIL-11R alone most probably cannot be ascribed to copurified IL-11 or other impurities in the soluble receptor preparations. Due to its trophic effects on motor neurons in vitro and in vivo, the neuropoietic cytokine CNTF is discussed as a drug to treat human motor neuron disease w7,16x. The observations that CT-1 and IL-11 plus soluble IL-11R also act as neurotrophic factors therefore suggest that the application of CT-1 or IL-11 might be a further therapeutic option to treat motor or sensory neurons affected by neuropathy. Moreover, a combined application of several neuropoietic cytokines or combinations with neurotrophins might be an option to overcome side effects induced by the application of one cytokine at high doses.
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Acknowledgements We thank Dr. D. Suter and Dr. J. Weissenberger for critically reading the manuscript. Support: IZKF CNS of the RWTH Aachen. This study was presented as part of the doctoral thesis of M. Thier ŽRWTH Aachen, 1998..
References w1x A. Acheson, J.C. Conover, J.P. Fandl, T.M. DeChiara, M. Russell, A. Thadani, S.P. Squinto, G.D. Yancopoulos, R.M. Lindsay, A BDNF autocrine loop in adult sensory neurons prevents cell death, Nature 374 Ž1995. 450–453. w2x H. Baumann, Y. Wang, K.K. Morella, C.-F. Lai, H. Dams, D.J. Hilton, R.G. Hawley, A. Mackiewicz, Complex of the soluble IL-11 receptor and IL-11 acts as IL-6-type cytokine in hepatic and nonhepatic cells, J. Immunol. 157 Ž1996. 284–290. w3x S.S. Cheema, L. Richards, M. Murphy, P.F. Bartlett, Leukemia inhibitory factor prevents the death of axotomised sensory neurons in the dorsal root ganglia of the neonatal rat, J. Neurosci. Res. 37 Ž1994. 213–218. w4x M.-J. Fann, P.H. Patterson, Neuropoietic cytokines and activin A differentially regulate the phenotype of cultured sympathetic neurons, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 43–47. w5x D.J. Hilton, A.A. Hilton, A. Raicevic, S. Rakar, M. Harrison-Smith, N.M. Gough, C.G. Begley, D. Metcalf, N.A. Nicola, T.A. Willson, Cloning of a murine IL-11 receptor Ž-chain: requirement of gp130 for high affinity binding and signal transduction, EMBO J. 13 Ž1994. 4765–4775. w6x J. Karow, K.R. Hudson, M.A. Hall, A.B. Vernallis, J.A. Taylor, A. Gossler, J.K. Heath, Mediation of interleukin-11-dependent biological responses by a soluble form of the interleukin-11 receptor, Biochem. J. 318 Ž1996. 489–495. w7x R.M. Lindsay, Therapeutic potential of the neurotrophins and neurotrophin-CNTF combinations in peripheral neuropathies and motor neuron disease, in: Growth Factors as Drugs for Neurological and Sensory Disorders. Ciba Found. Symp., Vol. 196, Wiley, Chichester, 1996, pp. 39–53. w8x M.F. Mehler, R. Rozental, M. Dougherty, D.C. Spray, J.A. Kessler, Cytokine regulation of neuronal differentiation of hippocampal progenitor cells, Nature 362 Ž1993. 62–65. w9x P.K. Mulderry, Neuropeptide expression by newborn and adult rat sensory neurons in culture: effects of nerve growth factor and other neurotrophic factors, Neuroscience 59 Ž1994. 673–688. w10x M. Murphy, K. Reid, M.A. Brown, P.F. Bartlett, Involvement of leukemia inhibitory factor and nerve growth factor in the development of dorsal root ganglion neurons, Development 117 Ž1993. 1173–1182. w11x P. Neddermann, R. Graziani, G. Ciliberto, G. Paonessa, Functional expression of soluble human interleukin-11 ŽIL-11. receptor and stoichiometry of in vitro IL-11 receptor complexes with gp130, J. Biol. Chem. 271 Ž1996. 30986–30991. w12x H. Neuhaus, B. Bettenhausen, P. Bilinski, D. Simon-Chazottes, J.-L. Guenet, A. Gossler, Etl2, a novel putative type-I cytokine receptor ´ expressed during mouse embryogenesis at high levels in skin and cells with skeletogenic potential, Dev. Biol. 166 Ž1994. 531–542. w13x D. Pennica, K.L. King, K.J. Shaw, E. Luis, J. Rullamas, S.-M. Luoh, W.C. Darbonne, D.S. Knutzon, R. Yen, K.R. Chien, J.B. Baker, W.I. Wood, Expression cloning of cardiotrophin 1, a cytokine that induces cardiac myocyte hypertrophy, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 1142–1146. w14x D. Pennica, K.J. Shaw, T.A. Swanson, M.W. Moore, D.L. Shelton, K.A. Zioncheck, A. Rosenthal, T. Taga, N.F. Paoni, W.I. Wood, Cardiotrophin-1, J. Biol. Chem. 270 Ž1995. 10915–10922.
84
M. Thier et al.r Molecular Brain Research 64 (1999) 80–84
w15x D. Pennica, V. Arce, T.A. Swanson, R. Vejsada, R.A. Pollock, M. Armanini, K. Dudley, H.S. Phillips, A. Rosenthal, A.C. Kato, C.E. Henderson, Cardiotrophin-1, a cytokine present in embryonic muscle, supports long-term survival of spinal motoneurons, Neuron 17 Ž1996. 63–74. w16x M. Sendtner, P. Carroll, B. Holtmann, R.A. Hughes, H. Thoenen, Ciliary neurotrophic factor, J. Neurobiol. 25 Ž1994. 1436–1453. w17x Z. Sheng, D. Pennica, W.I. Wood, K.R. Chien, Cardiotrophin displays early expression in the murine heart tube and promotes cardiac myocyte survival, Development 122 Ž1996. 419–428. w18x R. Simon, M. Thier, A. Kruttgen, S. Rose-John, O. Weiergraber, ¨ ¨
P.C. Heinrich, J.M. Schroder, J. Weis, Human CNTF and related ¨ cytokines: effects on DRG neurone survival, Neuroreport 7 Ž1995. 153–157. w19x T. Taga, T. Kishimoto, Gp130 and the interleukin-6 family of cytokines, Annu. Rev. Immunol. 15 Ž1997. 797–819. w20x M. Thier, R. Simon, A. Kruttgen, S. Rose-John, P.C. Heinrich, J.M. ¨ Schroder, J. Weis, Site-directed mutagenesis of human CNTF: func¨ tional analysis of recombinant variants, J. Neurosci. Res. 40 Ž1995. 826–835. w21x Y.-C. Yang, Interleukin 11: an overview, Stem Cells 11 Ž1993. 474–486.