The function of the soluble IL-6 receptor in vivo

immunology letters ELSEVIER

Immunology Letters 54 (1996) 177 184

The function of the soluble IL-6 receptor in vivo Malte Peters, Karl-Hermann Meyer zum Bi.ischenfelde, Stefan Rose-John* Department of Medicine, Division of Pathophysiology, Johannes Gutenberg-University of Main=, Obere Zahlhacher Strab'e 63. D-55 101 Mainz, Germany

Abstract

Interleukin-6 (IL-6) is considered an important mediator of acute inflammatory responses. Moreover. IL-6 functions as a differentiation and growth factor of hematopoietic precursor cells, B-cells, T-cells, keratinocytes, neuronal cells, osteoclasts and endothelial cells. IL-6 exhibits its action via a receptor complex consisting of a specific 1L-6 receptor (IL-6R) and a signal-transducing subunit (gpl30). Soluble forms of both receptor components are generated by shedding and are found in patients with various diseases such as AIDS, rheumatoid arthritis and others. The function of the soluble IL-6R in vivo is unknown. To discriminate between the biologic function of hlL-6 alone and that of the hlL-6/hslL-6R complex, mice transgenic for human IL-6, for the human soluble IL-6R and for both, human IL-6 and the human soluble IL-6R were analyzed and compared with nontransgenic littermates. While IL-6 transgenic mice exhibit elevated acute phase protein levels and develop plasmacytomas, hslL-6R single transgenic mice are hypersensitized towards human IL-6, mounting an acute phase protein gene induction at significantly lower IL-6 dosages compared to control animals. Furthermore, in hslL-6R transgenic mice, the acute phase response persists for a longer period of time and the IL-6 plasma half life was markedly prolonged. IL-6,slI-6R mice. however, develop massive hepatosplenomegaly caused by extramedullary hematopoisis in these organs. In IL-6- and IL-6R-single transgenic mice, no such effects were observed. Our study discloses a novel biologic effect of the hlL-6/hslL-6R complex, which is clearly distinct from that of hlL-6 alone. We provide evidence that the activation of the gpl30 signal transducer represents a major stimulation of growth and differentiation of hematopoietic progenitor cells. © 1996 Elsevier Science B.V. All rights reserved

Keyword~:Interleukin-6; Inflammatory responses; hIL-6/hslL-6R complex

1. Introduction

Interleukin 6 (IL-6) is a multi-functional cytokine and plays a central role as a differentiation and growth factor of hematopoietic precursor cells, B cells, T cells, keratinocytes, neuronal cells, osteoclasts and endothelial cells (reviewed in [1,2]). Moreover, IL-6 modulates the transcription of several liver-specific genes during acute inflammatory states [3]. Altered IL-6 serum levels have been described in several hematological diseases like plasmacytoma or Castleman's Disease [4,5], mesangial glomerulonephritis [6], osteoporosis [7], cachexia [8], rheumatoid arthritis [9], sepsis [10,11] and A I D S

[12,13]. * Corresponding author. Tel.: +49 6131 173363; fax: +49 6131 173364; e-mail: [email protected] 0165-2478/96/$12.00 © 1996 Elsevier Science B.V. All rights reserved PII S 0 1 6 5 - 2 4 7 8 ( 9 6 ) 0 2 6 6 9 - 7

IL-6 exhibits its action on target cells by acting through a receptor complex consisting of a specific IL-6-binding protein (IL-6R) and a signal-transducing subunit (gpl30). The IL-6/IL-6R complex induces the homodimerization of two gp130 molecules leading to a number of intracellular signaling events including activation of the transcription factor NF-IL-6, probably via the ras-microtubulus-associated protein (MAP) kinase cascade [14] and activation of the J a k / S T A T signaling pathway [15 17]. Soluble forms of the IL-6R (slL-6R) and gpl30 (sgpl30) are found in different body fluids and their concentrations m a y be elevated in patients with various inflammatory diseases such as A I D S and rheumatoid arthritis [13,18-20]. They are generated by limited proteolysis and are released from the cell surface [21 23]. It has also been demonstrated that an IL-6R m R N A

M. Peters et al./ Immunology Letters 54 (1996) 177 184

178

lacking the coding sequence for a transmembrane domain exists. This m R N A presumably derives from a differential splicing event and can lead to the synthesis of a soluble IL-6R protein [24]. The slL-6R retains its ligand-binding capacity [25,26] and associates with sgpl30 in the presence of IL-6 [23,27]. In vitro experiments have demonstrated that in contrast to other soluble receptors, such as the receptors for IL-1 or T N F , which are known to inhibit the effects of their ligands [28], slL-6R acts agonistically on cells that express gpl30. Most recently, it was shown that heart muscle cells respond to IL-6 only in the presence of the slL-6R [29]. By this trans-signaling pathway, slL-6R is able to activate target cells which express only gpl30 molecules on their cell surface but lack membranebound IL-6R (Fig. 1) [28,30]. Despite the in vitro studies cited above and the finding that soluble forms of IL-6R and gpl30 are found in various diseases, the function of these soluble proteins in vivo is not understood. Based on the unidirectional species specificity of IL-6 (Fig. 2) we have generated transgenic mice which express a soluble form of the human IL-6R. Since the endogenous murine IL-6 does not activate the human IL-6R, the mice develop no phenotype. Upon injection of human IL-6 in transgenic mice and nontransgenic littermates, the influence of the slL-6R on IL-6 specific responses can be identified. Crossing these mice with mice transgenic for human IL-6 resulted in double transgenic animals expressing human IL-6 and human slL-6R. Analysis of these transgenic animals allows the dissection of the biologic function of a cytokine alone from that of a cytokine/soluble receptor complex. We provide evidence that the activation of the gp 130 signal transducer represents a major stimulation of growth and differentiation of hematopoietic progenitor cells.

s,.-0.(t,

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human IL-6

murine IL-6R

human IL-6R

Fig. 2. Species specificityof human and murine IL-6. 2. Materials and methods

2.1. Chemicals Protein A Sepharose CL-4B was obtained from Pharmacia (Freiburg, Germany). D M E M , MEM and Penicillin/Streptomycin were obtained from Gibco (Eggenstein, Germany). FCS was obtained from Seromed (Berlin, Germany). The monospecific antiserum against IL-6R was prepared as described previously [26]. Recombinant human (rh) and recombinant murine (rm) IL-6 were prepared as previously described [31,32]; [c~-32p]dATP (110 TBq/mmol) was purchased from Amersham (UK). The anti-IL-6R mAB MT18 was supplied by Dr K. Yasukawa (TOSOH, Tokyo, Japan). The rabbit IgG anti-mouse antibody was obtained from Dako (Hamburg, Germany). The human IL-6 ELISA kit was obtained from CLB (Amsterdam, Netherlands). The human IL-6R ELISA kit was from Seromed (GieBen, Germany).

2.2. Generation of PEPCK/hIL-6 transgenic mice

I eleaseof the solubleIL-6R gp130

murine IL-6

gp130

Signal Fig. 1. Trans-signaling of soluble receptors of the IL-6 family.The soluble IL-6R (slL-6R) is generated by limited proteolysis by a presumably membrane-bound protease and released into circulation. The slL-6R captures an IL-6 molecule and binds to cells expressing only gpl30 on their surface, thereby initiating the intracellular signaling cascade.

A 1.1 kb c D N A fragment coding for the extracellular part of hIL-6R was amplified by the polymerase chain reaction and cloned into the Sail site of the pTZ18R vector. A 530 bp EcoRI-BglII D N A fragment of the mouse P E P C K promoter [33] was cloned upstream from the hIL-6R cDNA. A BamHI site was inserted upstream from the P E P C K promotor. A 1.2 kb D N A fragment corresponding to the 3' portion of the human fl-globin gene [34] followed by a poly-A site was inserted downstream of the hlL-6R c D N A between the Sail and the HindlII sites of the vector. The BamHIBamHI fragment containing the entire construct (Fig. 3) was gel purified and microinjected into the pronuclei

M. Peters et al./ Immunology Letters 54 (/996) 177 184

PEPCKPROMOTER

cDNA OF HUMAN

SOLUBLE IL-6R

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Fig. 3. PEPCK/hslL-6R construct injected into fertilized mouse eggs.

of fertilized eggs of NMRI and B602F1 mice. Mice were screened for the presence of hlL-6R by Southern blot of EcoRI-HindIII-digested tail DNA.

a 1.2 PstI-XhoI restriction fragment of human IL-6R cDNA, were used as probes. 2.6. Serum IL-6 measurements and cell growth

2.3. Generation of hlL-6/hslL-6R double transgenic mice Hemizygous double-transgenic mice coexpressing hlL-6 and hslL-6R (hlL-6/hslL-6R mice) were generated by crossing homozygous hslL-6R and hlL-6 [35] mice. The hlL-6 transgene was driven by the metallothionein promoter. Serum-concentration of hlL-6 ranged between 10-20 ng/ml in hlL-6, and hlL-6/hslL-6R mice, concentrations of hslL-6R ranged between 4-8 /~g/ml in hslL-6R, and hlL-6/hslL-6R mice (data not shown). At ages indicated in the figures, three mice were sacrificed and liver and spleen weights recorded. 2.4. Animal treatment Mice were maintained in a 12 h light-dark cycle under standard conditions and were provided food and water ad libitum. Procedures involving animals and their care were conducted in conformity with national and international laws and policies. Recombinant human IL-6 was injected intraperitoneally at doses indicated in Figs. 1-8. 2.5. RNA expression Mice were killed by cervical dislocation and RNA was isolated from different organs by the phenol/chloroform method [36] and subjected to Northern Blot analysis. Heat-denatured (5/~g) RNA per sample were fractionated on a 1% agarose gel with 7% formaldehyde. The separated RNA was transferred to GenScreen Plus membranes (Dupont-New England Nuclear, Dreieich, Germany) according to the supplier's instructions. The filters were prehybridized at 68°C for 2h in 10% dextran sulfate, 1 M NaCI, 1% SDS and hybridized in the same solution with [32P]cDNA fragments labeled by random priming [37]. A 0.9 kb HinfII restriction fragment of human Haptoglobin cDNA and

Blood was drawn by cardiac punction under general anesthesia of the mice. The human IL-6 ELISA was performed according to the supplier's instructions. Biologically active IL-6 was measured using the IL-6 dependent cell line B9 [38].

3. Results

3.I. Generation of PEPCK/hsIL-6R transgenic mice To drive expression of the human soluble IL-6R in the liver of transgenic mice, we used the promoter of the mouse PEPCK gene, which is neonatal and thereby permits to evaluate the influence of gene products after birth [33]. The promoter has been shown to be specific for liver and kidney [39]. DNA (Fig. 3) was microinjected into the pronuclei of fertilized eggs from NMRI mice. Four PEPCK/hsIL-6R transgenic founders were obtained and verified by Southern blot analysis. Mice were interbred to obtain homozygous and heterozygous transgenic PEPCK/hsIL-6R mice. 3.2. Characterization of PEPCK/hslL-6R transgenic mice Tail DNA was digested with EcoRl/Hindlll and used for Southern blot analysis. In homozygous mice, the level of the transgene was two-fold of that observed in heterozygous mice (data not shown). The hsIL-6RmRNA levels in homozygous mice were 5-fold higher than those observed in heterozygous mice (data not shown). Accordingly, in immunoprecipitation experiments, the highest levels of hsIL-6R were detected in homozygous mice (data not shown). Transgenic mice expressed the shIL-6R in the low microgram range as determined by ELISA (data not shown). When Northern blot analysis was performed with RNA from differ-

180

M. Peters et al./ Immunology Letters 54 (1996) 177-184

ent mouse tissues, hslL-6R-mRNA was detected in the liver and kidney, but not in heart, muscle and brain (data not shown).

Hapt oglobin

3.3. The hslL-6R transgenic mice are hypersensitized towards the human IL-6-induced acute phase protein expression One major activity of IL-6 is the induction of the hepatic acute phase response [40]. We therefore analyzed the induction of the acute phase protein gene expression by human IL-6 in the liver of hslL-6R transgenic mice. Various dosages of human IL-6 were administered to the mice intraperitoneally and mice were killed after 4 h (Fig. 4). In the upper panel, the haptoglobin mRNA expression in control animals is demonstrated. The strongest haptoglobin expression was seen when 40 /zg human IL-6 were injected. In heterozygous hslL-6R transgenic mice (middle panel), the strongest haptoglobin expression was seen at a ten-fold lower dose (4 pg) compared to control animals. In homozygous hslL-6R mice (lower panel), a human IL-6-dosage of as low as 0.4 /lg induced a significant haptoglobin mRNA expression. These experiments clearly demonstrate that the hslL-6R renders transgenic mice more sensitive to human IL-6 in a dose dependent fashion. In contrast, when control, heterozygous and homozygous hslL-6R transgenic animals

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Fig. 5. Time course of the hepatic acute phase protein expression.Haptoglobin expression in the liver of control mice ( - / ), heterozygous ( - / + ), and homozygous ( + / + ) transgenic mice was analyzed. Mice were injected intraperitoneally with 8 #g human IL-6 and sacrificed after time periods as indicated in the figure. R N A was prepared from the liver and subjected to Northern blot analysis. Filters were hybridized with a 32p-labeled 0.9 kB HinflI restriction fragment of human haptoglobin cDNA.

were injected with 8/~g of murine IL-6 and analyzed 4 h after injection, an equal induction of the acute phase response was seen in all animals (data not shown). 3.4. In hslL-6R transgenic mice the IL-6-induced acute phase protein expression is markedly prolonged

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Fig. 4. Dose-response of the hepatic acute phase protein expression.Haptoglobin expression in the liver of control mice ( - / ), heterozygous ( - / + ), and homozygous ( + / + ) transgenic mice was analyzed. Mice were injected intraperitoneally with various dosages of human IL-6 as indicated in the figures. Mice were killed 4 h following injection and R N A was prepared from the liver and subjected to Northern blot analysis. Filters were hybridized with a 32p-labeled 0.9 kb Hinfll restriction fragment of human haptoglobin cDNA.

Time course experiments were performed with 8 ktg human IL-6 injected intraperitoneally (Fig. 5). In control mice (upper panel), the strongest haptoglobin mRNA expression was detected after 8 h and declined thereafter. In heterozygous (middle panel) mice the response was equally strong after 8 and 24 h and in homozygous (lower panel) hslL-6R transgenic mice, the expression of haptoglobin peaked at 24 h and was still pronounced after 48 h when compared to control animals (upper panel) and heterozygous transgenic mice (middle panel). 3.5. The soluble IL-6R functions as a carrier protein for IL-6 prolonging the serum half life of IL-6 To investigate the fate of human IL-6 injected into IL-6R transgenic mice, we determined IL-6 serum levels

M. Peters et al./ Immunology Letters 54 (1996) 177-184

using the human IL-6 ELISA (Fig. 6). IL-6 (8/~g) was injected intraperitoneally into normal, heterozygous and homozygous transgenic mice and IL-6 serum levels were determined 4 h after IL-6 administration. IL-6 levels were markedly elevated in heterozygous and even higher in homozygous hslL-6R transgenic mice when compared to control animals (Fig. 6).

3.6. The hlL-6/hslL-6R double transgenic mice develop massive hepatosplenomegaly caused by proliferation of hematopoietic progenitor cells

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To discriminate between the biologic function of hlL-6 alone and that of the hlL-6/hslL-6R complex, double-transgenic mice coexpressing hlL-6 and hslL6R (hlL-6/hslL-6R mice), were generated. The hlL-6/ hslL-6R mice were compared with hslL-6R and hlL-6 single transgenic mice and nontransgenic littermates. The first differences between the transgenic mouse lines were seen as soon as 4-6 weeks after birth. In hlL-6/ hslL-6R mice, a marked increase of liver and spleen weights relative to the total body weight was observed, at week 20, the liver weight (Fig. 7A) had duplicated and the spleen weight (Fig. 7B) increased by a factor of five compared to single transgenic and nontransgenic littermates. In the livers and spleens of hlL-6/hslL-6R mice, the number of hematopoietic foci increased dramatically with time. The increase in liver and spleen weight was caused by a marked proliferation of hematopoietic cells in both organs (data not shown), which was absent in any other parenchymal organ. No such effects were seen in mice transgenic for hlL-6 or hslL6R nor in nontransgenic littermates. These data clearly indicate that continuous gpl30 stimulation by the complex of IL-6 and slL-6R leads to massive extramedullary hematopoiesis in spleen and liver.

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weeks Fig. 7. Liver and Spleen weight in transgenic mice. Liver (A) and spleen (B) weight of hlL-6/hslL-6R, hlL-6, hlL-6R mice and nontransgenic littermates. At ages indicated in the tigures, three mice were sacrificed and liver and spleen weights recorded. Data are shown as a ratio of organ weight/body weight.

4. Discussion

1500"

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Fig. 6. Correlation of serum levels of human IL-6 and transgenic slL-6R expression. IL-6 serum levels in control mice (open bars), heterozygous (hatched bars) and homozygous (filled bars) transgenic mice were determined with a human IL-6 ELISA. Mice were injected with 8 /tg of human IL-6 intraperitoneally and were analyzed 4 h after injection.

To determine the role of the slL-6R in vivo, we made use of the species specifity of human IL-6, which acts on human and murine IL-6R, in contrast to murine IL-6 which acts only on murine IL-6R. Transgenic mice expressing the human soluble I L-6R were generated and the function of the hslL-6R was studied in vivo without the confounding action of endogenous murine IL-6. There are several important conclusions that arise from this study. Firstly, the slL-6R makes animals more sensitive to the cytokine IL-6. According to studies performed in cell culture, the slL-6R renders target cells more sensitive and enables cells to respond to I L-6 which by themselves do not bind the cytokine due to lack of surface expression of the membrane bound IL-6R (trans-signaling, Fig. 1) [18,28,30]. The degree of

182

M. Peters et al, / Immunology Letters 54 (1996) 177-184

sensitization we observed in the transgenic mice, however, exceeded by far the effect seen in cell culture, when human IL-6 was injected into hslL-6R transgenic animals and the IL-6-mediated acute phase protein expression was measured, these mice were found to be 10-100-fold more sensitive towards human IL-6 as compared to control animals. In contrast, the injection of murine IL-6 did not result in an increased acute phase protein expression in hslL-6R transgenic mice when compared to control animals. This finding confirms the species specificity of murine IL-6, which has no effect on human target cells (Fig. 2) and argues against any interference of endogenous murine IL-6 with the hslL-6R. Since the acute phase response induced by murine IL-6 was equal in control, heterozygous and homozygous hslL-6R transgenic animals, one can conclude that the binding of human IL-6 to membrane bound and soluble murine IL-6 receptor does not contribute to the dose-dependent IL-6-induced hypersensitization of hslL-6R transgenic mice demonstrated in our study. Two other members of the IL-6 family of cytokines, IL-11 and ciliary neurotrophic factor (CNTF) have been shown to bind to receptors highly homologous to the IL-6R. Consequently, it has been shown that soluble receptors for IL-11 and CNTF in complex with their cognate cytokines act as agonists on cells expressing gpl30 and the combination of gpl30 and LIF-R, respectively [41,42]. Therefore, it can be concluded that the biological function of the slL-6R defined in this study can also be ascribed to the soluble receptors for IL-11 and CNTF. Secondly, time course experiments revealed that in hslL-6R transgenic animals, the IL-6-mediated acute phase response persisted much longer as compared with control animals. In homozygous hslL-6R transgenic mice, the acute phase response persisted as long as 48 h. Thirdly, in hslL-6R transgenic animals, the IL-6 serum levels are markedly elevated and the plasma half life of IL-6 is significantly prolonged. In a dose-dependent fashion, hslL-6R transgenic mice have 2 3-fold higher IL-6 serum levels as compared to control animals. Moreover, in time course experiments (Fig. 5) IL-6 levels are detectable as long as 48 h in hslL-6R transgenic animals, whereas in control mice, IL-6 levels could be measured for only 24 h. Many sR (TNF-R, IL-1R, IL-4R, etc.) act antagonistically competing with the binding of the cytokines to membrane receptors [43 45]. Some agonistic cytokine/ sR-complexes, however, stimulate signal transducing receptors, rendering cells sensitive to the respective cytokine which lack specific cytokine receptors on their surface. We have developed a transgenic mouse model to define biologic functions of a cytokine alone, acting on specific membrane receptors, and the function of a cytokine/sR complex, acting on target cells lacking

specific receptors. Double-transgenic mice coexpressing human (h) IL-6 and the shlL-6R (hlL-6/hslL-6R mice), develop extensive extramedullary hematopoiesis. In hlL-6- and hslL-6R-single transgenic mice no such effect was observed. Therefore, the transgenic mouse model described here allows to discriminate between the biologic action of a cytokine alone and a cytokine/ soluble receptor-complex and may clarify the function of the growing number of soluble cytokine receptors [46]. Our study discloses a novel biologic effect of the hlL-6/hslL-6R complex, which is clearly distinct from that of hlL-6 alone. We provided evidence that the activation of the gpl30 signal transducer represents a major stimulation of growth and differentiation of HPCs. This notion is underscored by the greatly diminished number of these cells in gpl30 deficient mice [47]. Since IL-6 and IL-11 act via the gpl30 homodimer, loss of IL-6 can be complemented by IL-11 explaining why IL-6 deficient mice are viable and only have slightly decreased numbers of hematopoietic progenitor cells [48,49]. Our data strongly suggests a high expression of gpl30 and the absence of IL-6R on HPCs. In mice overexpressing cytokines requiring the gpl30/LIF-Rheterodimer for signal transduction, like LIF [50] or OSM [51], hematopoietic effects are scarce, indicating that hematopoietic progenitor cells express little or no LIF-R on their surface. In summary, the transgenic mouse model described in this study permits to discriminate between IL-6 responsive and unresponsive cells (Fig. 8). Cells expressing the IL-6R on their surface are responsive towards the IL-6/IL-6R complex and IL-6 alone (Fig. 8, left panel). Cells which express only gpl30 signal transducing molecules on their surface but lack the B

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IL-6

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3& IL-6-responsive cell e.g. hepatocyte, B-cell

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Fig. 8. IL-6-responsiveand lL-6-unresponsivecells. (A) Cellsexpressing the membrane bound IL-6R on their surface respond to IL-6 alone and to the IL-6/IL-6Rcomplex.On thesecells,the slL-6Rleads to sensitization and prolongation of the IL-6 response. (B) Cells expressing only the gpl30 signal transducer on their surface but not the IL-6R, respond only to the IL-6/IL-6Rcomplex via trans-signaling.

M. Peters et al./ Immunology Letters 54 (1996) 177 184

specific IL-6R, are responsive only towards the IL-6/IL6R complex and not towards IL-6 alone. We have clearly identified hematopoietic progenitor cells to be IL-6 unresponsive. However, in the presence of the IL-6/IL-6R complex these cells may be significantly activated.

Acknowledgements We thank Dr M. Blessing (University of Mainz, Germany) for continuous support and advice during the study. Dr B. Samols (Cleveland, USA) is thanked for the gift of the PEPCK promoter. The help of Dr R. Kluge (RWTH Aachen, Germany) during the initial phase of the study is gratefully acknowledged. We are indebted to M. Fischer for the preparation of rhIL-6 and to S. Saya for help with the artwork. This work was supported by the Deutsche Forschungs-Gemeinschaft (Bonn, Germany) and the NMFZ (Mainz, Germany).

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