Stem Cells, Vol. 15, No. 2, 119-124,
March 1997
© 1997 AlphaMed Press
Expression of Murine Interleukin 11 and its Receptor 
-Chain in Adult and Embryonic Tissues
Alan J. Davidsona,
Sally-Anne Freemana,
Kathryn E. Crosiera,
Clive R. Woodb,
Philip S. Crosiera
a Department of Molecular Medicine, School of Medicine, University of Auckland, Auckland, New Zealand;
b Genetics Institute, Inc., Cambridge, Massachusetts, USA
Key Words. Interleukin 11 • ES cells • IL-11 receptor 
-chain • Embryonic development
Dr. Philip Crosier, Department of Molecular Medicine, School of Medicine, University of Auckland, Private Bag 92019, Auckland, New Zealand.
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Abstract
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Interleukin 11 (IL-11) is a multifunctional cytokine that has diverse effects on blood cells and their precursors and on a number of cell types outside of the hematopoietic system. The cDNAs encoding murine IL-11 and its receptor -chain (IL-11R) have recently been isolated. We have used the RNase protection assay to examine the expression of murine IL-11 and IL-11R in a range of adult mouse tissues, in embryos, and during development of embryonic stem (ES) cells into cystic embryoid bodies in vitro. The testis showed a high level of IL-11 gene expression while a much lower level of expression was detected in the lung, stomach, small intestine, and large intestine. Expression of IL-11 was not detected between day 10.5 and day 18.5 post coitum of embryonic development or in differentiating ES cells in vitro. In contrast, the IL-11R was found to be expressed in all adult tissues examined, during embryonic development, and in totipotent and differentiating ES cells.
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Introduction
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Interleukin 11 (IL-11) is a pleiotropic cytokine that was initially identified as a soluble factor produced by PU-34 primate bone marrow stromal cells [1]. IL-11, acting either alone or in synergy with other cytokines, is capable of stimulating megakaryopoiesis, thrombopoiesis, myelopoiesis and lymphopoiesis in vitro [2]. Other activities of IL-11 outside of the hematopoietic system include inhibition of adipogenesis [3, 4], induction of acute phase proteins [5, 6], induction of neuronal differentiation [7], and as a potential regulator of cartilage and bone function [8, 9]. IL-11 has also been found to have a protective effect on the gastrointestinal mucosa following cytoablative treatments [10, 11]. The murine IL-11 cDNA was cloned from a lipopolysaccharide-stimulated fetal thymic cell line (T2) that was generated by infecting day 14 post coitum (pc) fetal thymic lobes from C57BL/10J mice with a retrovirus encoding v-Ha-ras and v-mycmc29 oncogenes [12]. The biological activity of murine IL-11, as assessed by its ability to stimulate the proliferation of the T10 plasmacytoma cell line and its actions on primary murine hematopoietic cells, is indistinguishable from that of human IL-11 [13].
IL-11, interleukin 6 (IL-6), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and oncostatin M belong to a group of cytokines that utilize the common signal-transducing receptor subunit gp130. The overlapping biological function of these cytokines can be attributed, at least partly, to the sharing of this common transducing component in their high-affinity receptor complexes [14-16]. Ligand-binding specificity is conferred by cytokine-specific -chains [17]. The cDNA encoding the -chain component of the murine IL-11 receptor complex has been isolated [18, 19]. The extracellular domain of the IL-11R contains the structural features typical of the hematopoietin receptor family, including four conserved cysteine residues and the five-amino-acid motif WSXWS. Overall, the IL-11R is most similar to the IL-6 and CNTF receptor -chains. High-affinity IL-11 binding is only achieved following coexpression of gp130 and the IL-11R [18].
Although considerable effort has gone into elucidating the physiological actions of IL-11, relatively little is known about in vivo sites of expression of both IL-11 and its receptor -chain. In the present study, we have used the RNase protection assay to analyze the sites of expression of the genes encoding murine IL-11 and its receptor -chain. IL-11 gene expression was detected in adult testis, lung, stomach, small intestine, large intestine, and primary fibroblasts. The IL-11R was found to be expressed in all adult tissues examined, as well as in totipotent and differentiating embryonic stem (ES) cells and whole embryos between days 10.5 and 18.5 pc.
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Materials and Methods
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Cell Culture
The ESD3 embryonic stem cell line [20] was maintained on gelatin-coated dishes in Dulbecco's modified Eagle's medium (DMEM) with additives according to established procedures [21], in the presence of LIF. Cystic embryoid bodies were established following collagenase treatment of ES cells and subsequent suspension culture in bacteriological-grade petri dishes in DMEM with additives in the absence of LIF [22]. Proliferating cultures of adult mouse lung fibroblasts were established from fresh tissue samples. The cultures of lung fibroblasts and the cell line T2 were maintained in DMEM and RPMI 1640 media respectively, containing 10% fetal calf serum, 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine and 50 µM 2-mercaptoethanol. The Leydig (TM3) and Sertoli (TM4) cell lines were obtained from the American Type Culture Collection and maintained in DMEM containing 10% fetal calf serum and the same additives outlined above. The culture characteristics of the TM3 and TM4 cell strains are consistent with those seen in primary cultures of Leydig and Sertoli cells, respectively [23].
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Mice
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c57BL/6J mouse stocks obtained from Jackson Laboratories (Bar Harbor, ME) were bred at the University of Auckland School of Medicine Animal Facility (Auckland, New Zealand). The detection of a vaginal plug the first morning after mating was defined as embryonic day 0.5. Adult mouse tissues were obtained from C57BL/6J mice.
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RNA Isolation
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Total RNA was prepared from cell lines, embryoid bodies, and adult and embryonic mouse tissues using the acid guanidinium isothiocyanate method [24]. Total RNA from ES cells maintained in the presence of LIF was only extracted from undifferentiated cultures as assessed by phase-contrast microscopy. Clone 31A, encoding the full-length murine IL-11 cDNA [13], was subcloned into the pcDNA3 expression vector (Invitrogen Corp.; San Diego, CA) and transiently transfected into COS cells using the diethylaminoethyl dextran method [25]. Total RNA from these transfected COS cells was used as a positive control.
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Ribonuclease Protection Assay
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Total RNA (10-50 µg) was hybridized to 32P-labeled RNA probes encoding sequences of either murine IL-11 or the murine IL-11R and ß-actin, as described previously [26]. The amount of total RNA used in each assay is specified in the corresponding figure legend. Total RNA (0.5 µg) from COS cells transiently transfected with the full-length murine IL-11 cDNA and total RNA (20 µg) from T2 cells were used as positive controls for assays of IL-11 gene expression. Total RNA (10 µg) from day 12.5 pc mouse embryos was used as a positive control for assays of IL-11R gene expression. As a negative control, yeast tRNA was used. The probe used for the analysis of IL-11 expression was derived from a Xho I-Apa I fragment encoding nucleotides (nt) 144 to 526 of the full-length murine IL-11 cDNA [13]. The probe used for the analysis of IL-11R expression was derived from a Hind III-Rsa I fragment encoding nucleotides 84 to 390 of the full-length IL-11R cDNA [18]. A partial cDNA sequence for the IL-11R that was generated by the polymerase chain reaction was kindly provided by Jim Tobin (Genetics Institute; Cambridge, MA). The sequences encoding both of these probes were subcloned into the pBluescript SK- vector (Stratagene; La Jolla, CA) and 32P-labeled antisense riboprobes synthesized using T7 RNA polymerase for the IL-11-specific probe and T3 RNA polymerase for the IL-11R-specific probe. The length of the IL-11-specific probe was 470 nt, and IL-11 transcripts protected a fragment of 383 nt. The length of the IL-11R probe was 405 nt, and the protected fragment was 307 nt in length. A riboprobe was also constructed from a Sal I-Sma I fragment of the human ß-actin cDNA. The length of the ß-actin probe was 132 nt, and ß-actin transcripts protected a fragment of 54 nt.
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Results
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Expression of IL-11
The expression of IL-11 in a range of adult and fetal mouse tissues and cell lines was examined using the RNase protection assay (Fig. 1
). As positive controls, IL-11 mRNA was assayed in RNA extracted from the T2 cell line from which the murine IL-11 cDNA was isolated and from COS cells transiently transfected with a murine IL-11 cDNA expression plasmid. IL-11 gene expression was not detected in adult brain, salivary gland, thymus, heart, liver, spleen, esophagus, adrenal gland, kidney, bladder, uterus, ovary, skeletal muscle, or bone marrow (data not shown). The adult testis was the only tissue where a high level of IL-11 gene expression was found, as indicated by the presence of a protected fragment of 383 nt after exposure of the autoradiograph for 16 hours. To investigate this expression further, the Leydig (TM3) and Sertoli (TM4) cell lines were examined. These are clonal cell lines derived from the normal testes of immature BALB/c nu/+ mice [23]. IL-11 expression was not detected in these cell lines. A readily detectable level of IL-11 gene expression was found in the lung and a lower level detected in the stomach and intestines. The latter signals were just detectable after exposure of the autoradiographs for 6-8 days. Although the expression in the lung was low, moderate levels were detected in proliferating cultures of adult mouse lung fibroblasts. The expression of IL-11 was not detected in totipotent or differentiating ES cells or in whole embryos at days 10.5 through 18.5 pc (data not shown).
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Figure 1. Expression of IL-11 in murine lung fibroblasts, cell lines, and adult C57BL/6J mouse tissues. RNase protection analysis was performed on total RNA (20 µg) from proliferating lung fibroblasts (lung fb), the Leydig (TM3) and Sertoli (TM4) cell lines, and the tissues lung, stomach, small intestine (int), large intestine, and testis. As positive controls, total RNA (0.5 µg) from COS cells transfected with the full-length murine IL-11 cDNA and total RNA (20 µg) from T2 cells were used. As a negative control, tRNA (20 µg) was used. The autoradiographs were exposed for 6-8 days for samples lung fb, lung, stomach, small intestine, and large intestine, and 16 hours for samples COS control, T2, TM3, TM4, and testis. The size of the IL-11 probe was 470 nt. The protected fragment representing the presence of IL-11 transcripts was 383 nt in length. The ß-actin probe was 132 nt in length and the 54 nt ß-actin protected fragment is shown in each lane as an RNA-loading control. The markers were pBR322 digested with Msp I.
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Expression of the il-11 Receptor
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The expression of IL-11R in a range of adult mouse tissues was indicated by the presence of a 307 nt protected band. A less intense 290 nt band that was not represented in the tRNA control was consistently observed below the 307 nt band and was regarded as a partial degradation product of the 307 nt protected fragment. Additional bands, including the one migrating at approximately 405 nt, are observed in all RNA samples including the tRNA control and are regarded as nonspecific. Expression of IL-11R was relatively high in the brain, salivary gland, heart, liver, adrenal gland, and kidney, while a moderate level of expression was detected in the lung, esophagus, bladder, ovary, testis, skeletal muscle, and day 14 pc placenta. Low levels of IL-11R expression were found in the spleen, bone marrow, stomach, and uterus (Fig. 2). A lower level of expression was found in the thymus, small intestine, and large intestine in which a larger amount of RNA was used in the assay in order to obtain a comparable signal.
The finding that IL-11 was expressed in the testis, the T2 cell line, and in lung fibroblasts prompted us to examine the expression of IL-11R in the TM3, TM4, and T2 cell lines and in proliferating mouse lung fibroblasts. IL-11R transcripts were detected in the murine Leydig (TM3) and Sertoli (TM4) cell lines, in the T2 cell line, and in proliferating adult mouse lung fibroblasts (Fig. 3). A low level of the 307 nt-protected fragment was detected in the TM3 cell line. However, two smaller protected fragments that were not represented in the tRNA control were detected migrating at approximately 115 and 160 nt, respectively. These two fragments were also detected at lower levels in the TM4 cell line (Fig. 3), skeletal muscle, and adrenal gland, while higher levels were found in the bladder (data not shown). Further work is needed to determine whether these fragments are due to alternative splicing of IL-11R transcripts.
The expression of IL-11R was examined in totipotent ES cells growing in LIF (termed "day 0") and in differentiating cystic embryoid bodies at days 6, 12, and 18 following LIF withdrawal (Fig. 4A). Totipotent ES cells expressed a low level of IL-11R. Expression of the receptor -chain was found to increase as the embryoid bodies differentiated, such that the highest level of expression was found at day 18. Abundant transcripts for the IL-11R were found in total RNA extracted from whole embryos at embryonic days 10.5, 12.5, 14.5, 16.5, and 18.5 pc. A uniform level of receptor expression was found during these stages of development (Fig. 4B).
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Discussion
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We have used sensitive RNase protection assays to survey the expression of IL-11 and its hematopoietin receptor -chain in normal murine tissues and cell lines. IL-11 expression was found in a restricted set of tissues. In contrast, expression of the IL-11R was found in a wide range of adult tissues. The signaling component of the IL-11 receptor complex, gp130, has also been reported to be widely expressed in adult murine tissues. However, whereas IL-11R gene expression was found to remain at high levels in embryonic development from day 10.5 to 18.5 pc, the expression of gp130 has been reported to decrease in this time period [27]. The highest level of IL-11 mRNA was detected in the testis with lower levels in the lungs, stomach, small intestine, and large intestine. The expression of IL-11 in the testis, combined with our detection of IL-11R mRNA in the Leydig (TM3) and Sertoli (TM4) cell lines, indicates a role for this cytokine in testis function. This conclusion is supported by the recent report of IL-11 expression in round spermatids in stage VI-IX seminiferous tubules [28].
A number of lines of evidence indicate an important role of IL-11 in epithelial tissue biology. These data include the induction of IL-11 expression in lung epithelial cells by IL-1, and transforming growth factor-ß, and by infection with respiratory syncytial virus [29], and demonstrations that IL-11 has a protective effect on the gastrointestinal mucosa following chemotherapy or irradiation [10, 11]. Consequently, the finding that IL-11 is expressed in the lung and gut is consistent with our growing knowledge of the epithelial role of this cytokine. Furthermore, our detection of IL-11 and IL-11R mRNA in primary murine lung fibroblasts suggests that this cytokine may act in an autocrine or paracrine manner.
The differentiation of ES cells in vitro into embryoid bodies containing blood islands has been used as a model system for studying early mouse hematopoiesis [20, 30, 31]. The addition of IL-11 to this model system increases the number of embryoid bodies and enhances the number of hematopoietic precursors that are generated at day 10 and day 14 of embryoid body development [31]. In agreement with these findings, expression of IL-11R was detected in totipotent ES cells and in embryoid bodies at days 6, 12, and 18 following LIF withdrawal. Furthermore, IL-11R is expressed in the yolk sac of day 8.5 pc embryos [19]. These findings suggest that IL-11 may be involved in regulating early embryonic blood development. However, IL-11 gene expression was not detected in totipotent ES cells, embryoid bodies, or whole embryos at days 10.5, 12.5, 14.5, 16.5, or 18.5 pc. This may be due to an extraembryonic source of IL-11. The chorion or placenta may provide the embryo with IL-11. Indeed, the human trophoblast cell line tPA-30-1 has been found to express IL-11 [1]. Alternatively, the level of IL-11 expression needed to elicit a physiological effect may be low, such that it was below the level of sensitivity of our assays. Further work will increase the sensitivity of our detection by using in situ hybridization. In addition, given the induction of IL-11 expression in vitro by cytokines such as IL-1 and transforming growth factor-ß [29, 32, 33], and the increased serum level of human IL-11 during acute thrombocytopenia [34], it is likely that local physiological changes can regulate IL-11 expression in vivo. Thus, it will be important to monitor IL-11 and IL-11R expression in various physiological states such as the stress hematopoiesis following 5-fluorouracil treatment or irradiation.
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Acknowledgments
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We are grateful to Jim Tobin (Genetics Institute; Cambridge, MA) for providing the plasmid pED-smIL11R from which the IL-11R probe was derived. This work was supported in part by a grant from the Auckland Medical Research Foundation, Auckland, New Zealand.
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accepted for publication December 4, 1996.
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Abstract
Introduction
