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Human interleukin-11 (hIL-11) is a pleiotropic cytokine that is involved in numerous biological activities such as hematopoiesis, osteoclastogenesis, neurogenesis and female fertility. IL-11 is obviously a key reagent to study the IL-11 receptors. However, conventional radio-iodination techniques lead to a loss of IL-11 bioactivity. Here, we report the construction and the production of a new recombinant human IL-11 (FP Delta IL-11). In this molecule, a specific phosphorylation site (RRASVA) has been introduced at the N-terminus of rhIL-11. It can be specifically phosphorylated by bovine heart protein kinase and accordingly, easily radiolabeled with (32)P. A high radiological specific activity (250,000 c.p.m x ng(-1) of protein) was obtained with the retention of full biological activity of the protein. The binding of (32)P-labeled FP Delta IL-11 to Ba/F3 cells stably transfected with plasmids encoding human IL-11 receptors alpha and beta chains (IL-11R alpha and gp130) was specific and saturable with a high affinity as determined from Scatchard plot analysis. Availability of this new ligand should prompt further studies on IL-11R structure, expression and regulation.

Anti-human interleukin-6 (human IL-6) and anti-human IL-6 receptor (IL-6R)-neutralising monoclonal antibodies (mAbs) are among the most promising human IL-6-specific inhibitors and have been shown to exert short-term beneficial effects in clinical trials. Simultaneous treatment with different anti-human IL-6 or anti-human IL-6R mAbs was recently suggested to be a potent way to inhibit the action of the cytokine in vivo. Although some of these mAbs are already used, their mechanisms of action and the location of their epitopes on the surface of human IL-6 and human IL-6R are still unknown. Here, we analysed the capacity of several anti-human IL-6 and anti-human IL-6R mAbs to inhibit the interaction between human IL-6, human IL-6R, and human glycoprotein 130 (gp130). We mapped the epitopes of several of these mAbs by studying their binding to human IL-6 and human IL-6R mutant proteins. Our results show that several anti-human IL-6 and anti-human IL-6R-neutralising mAbs block the binding between human IL-6 and human IL-6R, whereas others block the binding to gp130. We provide evidence that some of the latter mAbs inhibit interaction with gp130beta1, whereas others interfere with the binding to gp130beta2. Our results suggest that residues included in the C'D' loop of human IL-6R interact with gp130beta2.

Genomic DNA sequencing in the vicinity of the pstA-1 gene from Mycobacterium tuberculosis allowed us to clone, sequence and identify a gene encoding a 70-kDa protein. The size of the protein was confirmed by in vitro coupled transcription/translation. Its N-terminal domain shows extensive sequence similarity with the catalytic domain of eukaryotic serine/threonine protein kinases, and the protein was therefore called Mbk (mycobacterial protein kinase). The deduced amino acid sequence contains two transmembrane segments, which flank a highly repetitive region, suggesting a receptor-like anchoring. The mbk gene was overexpressed in Escherichia coli and the gene product (Mbk) was purified as a fusion protein with gluthatione S-transferase. Recombinant Mbk was found to be autophosphorylated on threonine residues and capable of phosphorylating myelin basic proteins from bovine brain and histones from calf thymus on serine residues, both in a manganese-dependent manner. The phosphorylation of myelin basic proteins by Mbk was inhibited by calcium and by staurosporine, a widely used inhibitor of eukaryotic protein serine/threonine kinases. A similar gene was found in Mycobacterium bovis BCG DNA by Southern blot analysis. Its expression was detected in cultures of M. bovis BCG by reverse transcriptase/PCR. Although its biological role is unknown, it is the first serine/threonine protein kinase characterized in Mycobacteria.

The alpha-subunit of interleukin-6 (IL-6) receptor is a member of the hematopoietin receptor family. The alignment of its amino acid sequence with those of other members of this family (human somatotropin receptor/murine IL-3 receptor beta and human IL-2 receptor beta) has suggested that amino acids included in two SSFY repeats found in each of its hematopoietin receptor domains, contribute to the binding of the ligand. The involvement of these amino acids in IL-6 binding and signal transduction was studied by site-directed mutagenesis and molecular modelling. We present a computer-derived three-dimensional model of the IL-6/IL-6 receptor complex based on the structure of the human somatotropin/human somatotropin receptor complex. This model allowed the location of distinct regions important for IL-6 and gp130 binding. We show that some of the residues included in the SSFY repeats located in our IL-6 receptor model in the loops between beta-strands E and F of domain-I and B' and C', of domain-II, participate in the formation of a major IL-6-binding site. These residues are necessary for IL-6 and gp130 binding and for signal transduction. Using our IL-6 receptor mutants we mapped the epitopes of our anti-(IL-6 receptor) neutralising monoclonal antibodies to these residues. Our results demonstrate that a generic hematopoietin receptor family structural module can be used for the study of both alpha and beta receptor subunits belonging to this family.

Interleukin 6 (IL-6) is an important mediator of various kinds of inflammatory and immune responses. The human amniotic cell line UAC has an increased number of IL-6 receptors after treatment by glucocorticoids. To find a possible activity of IL-6 on these cells, a cDNA library of IL-6- and dexamethasone-treated cells was screened with cDNA probes from both induced and non-induced cells. Two cDNAs showed a differential hybridization signal. The first one corresponds to metallothionein, a group of small cysteine-rich proteins thought to participate in the metabolism and storage of zinc and to protect cells against oxidative damage. A second cDNA corresponds to the recently cloned cDNA of band 7 integral membrane protein also called stomatin. In hereditary stomatocytosis, absence of this protein in erythrocyte membranes is associated with high Na+ and low K+ intracellular concentrations [Stewart, G. W., Hepworth-Jones, B. E., Keen, J. N., Dash, B. C. J., Argent, A. C. & Casimir, C. M. (1992) Blood 79, 1593-1601]. In UAC cells both metallothionein and stomatin are induced by dexamethasone and IL-6 in a more than additive manner. Western blot analysis shows that stomatin protein is induced in a similar way as its mRNA. IL-6 and dexamethasone induce a state of resistance against hydrogen peroxide toxicity in UAC cells. Metallothionein induction might be partly responsible for this cytoprotection against oxidative stress.

Three internal-amino acid deletions of amino acids 171-179 of human interleukin 6 (IL-6) were introduced at the cDNA level. While all deletion proteins were biologically inactive, immunoprecipitations with a set of conformation-specific anti-(IL-6) monoclonal antibodies showed that only mutant delta 177-179 does not present major alterations in folding. This finding, together with the observation that delta 177-179 is not able to compete with IL-6 for binding to the soluble human IL-6 receptor, suggested that some or all of these three residues participate to the composition of the receptor-binding site of human IL-6. A large number of single-amino-acid-substitution mutants were generated in residues 177, 178 and 179. Their detailed analysis revealed that Arg179 is crucial for activity in mouse cells, because all amino acid substitutions in this position cause a dramatic drop of biological activity on murine hybridoma cells without affecting the overall protein folding. The only substitution which preserved some residual activity was the conservative Arg to Lys change. This demonstrates the absolute requirement for a positive charge in position 179 for the interaction of human IL-6 with its receptor.

Iodinated recombinant human interleukin-6 (125I-rhIL-6) was intravenously injected into rats and its fate was studied during 24 h. Between 10-20 min after a single-dose injection, 125I-rhIL-6 accumulated in liver as previously reported [Castell et al. (1988) Eur. J. Biochem. 177, 357-361]. After 1 h, the radioactivity disappeared from the liver and accumulated in skin, reaching 35% of injected 125I-rhIL-6 5-8 h after injection. No comparable accumulation of radioactivity was found in skin when [125I]iodide or rat serum 125I-albumin was administered. Finally the radioactivity was detected as [125I]iodide in urine. Autoradiographic analysis of skin sections 5 h after 125I-rhIL-6 injection showed radioactivity in the interstitium. When the experiments were carried out with [35S]rhIL-6, essentially the same results were obtained: a decrease in radioactivity in the liver after 20 min, and a substantial increase in skin 7 h after injection. In vitro experiments showed that 125I-rhIL-6 is degraded by rat and human fibroblasts, whereas no degradation was observed with rat hepatoma cells (Fao) or human hepatocytes. These observations suggest the involvement of skin in the catabolism of IL-6.

When human fibroblast cells were stimulated with poly(I) X poly(C) in the presence of cycloheximide for the production of interferon-beta (IFN-beta), a 26-kDa protein could be immunoprecipitated by antiserum raised against partially purified human IFN-beta [Content, J., De Wit, L., Pierard, D., Derynck, R., De Clercq, E. & Fiers, W. (1982) Proc. Natl Acad. Sci. USA 79, 2768-2772]. In our hands this 26-kDa protein showed no antiviral activity. Other investigators have, however, reported the presence in the same conditions of a second type of IFN, a so-called beta 2 species [Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, L., Soreq, H., Nir, U., Wallach, D., Perricaudet, M., Tiollais, P. & Revel, M. (1980) Proc. Natl Acad. Sci. USA 77, 7152-7156] of which the mRNA structure and protein characteristics strongly suggests identity with the 26-kDa product. In this paper we describe the nucleotide sequence of the 26-kDa cDNA and part of the corresponding genomic clone. The cDNA clones were isolated from a library made with mRNA from induced human fibroblasts. As, however, the information thus obtained was still incomplete, genomic clones were isolated from a total human DNA library. In this way, the entire region coding for the 26-kDa protein was established, as well as the neighbouring sequences including the inducible promoter area. From the deduced polypeptide sequence a number of characteristics of the 26-kDa protein can be explained. It turns out that the 26-kDa protein gene and the so-called 'IFN-beta 2' gene are identical. However, extensive homology searches indicate that the 26-kDa protein does not show statistically significant sequence homology with any known interferon species. Hence, the question of whether the 26-kDa product represents a novel IFN species remains open.

2'-Phosphodiesterase activity was investigated, by measuring either the disappearance of (2',5')oligo(adenylate) or the release of 5'AMP, in several human cell lines (RSa, IFr, HEC-1, WGAr and HeLa) possessing different sensitivities to interferon, and treated or untreated with human interferon. In various cell lines whose (2'-5')oligo(adenylate) synthetase was normally induced by interferon treatment, both kinetic studies and measurements at different enzyme concentrations indicated that 2'-phosphodiesterase activity remained unchanged after interferon treatment. This observation was confirmed over a broad range of substrate concentrations (1-25000 nM). The activity of 2'-phosphodiesterase was dependent on Mg(OAc)2. Our results indicate that in various human cell lines the modulation of (2'-5')oligo(adenylate) metabolism by interferon does not involve an increase of 2'-phosphodiesterase activity.

A human-leukocyte-derived antiviral protein (22-kDa factor), known to be an inducer of interferon-beta (IFN-beta) in fibroblastoid cells, and to be closely related to interleukin-1 (IL-1), was shown to likewise act as inducer of the mRNA of a 26-kDa secreted protein. This protein was first described as the gene product of an mRNA that is co-induced with the mRNA of IFN-beta by superinduction of fibroblasts (treatment with dsRNA and cycloheximide). Subsequently it was shown to be induced by treatment with cycloheximide only. The 22-kDa factor induced high levels of the 26-kDa-protein mRNA and low levels of IFN-beta mRNA. Addition of cycloheximide to the 22-kDa factor resulted in further significant increases in mRNA levels for both the 26-kDa-protein and IFN-beta. These observations add to the evidence already available that transcription of the genes for IFN-beta and the 26-kDa-protein are differently regulated. The observation that a factor that belongs to the IL-1 family induces the 26-kDa-protein suggest that the latter plays a role as an intermediary or effector molecule in inflammatory or immunoregulatory processes.

Among the various proteins which are induced when human cells are treated with interferon, a predominant protein of unknown function, with molecular mass 56 kDa, has been observed. With the aim of exploring the molecular basis of the regulation of this protein and of its mRNA, in order to understand its biological function and its possible contribution to the various antiviral and non-antiviral actions exerted by interferons, we cloned a full-length cDNA copy of the 1.8-1.9 X 10(3)-base 56-kDa-protein mRNA and determined its sequence. The cDNA contains 1662 nucleotides derived from the 56-kDa-protein mRNA, including a poly(A) tail of 20 residues. Primer extension experiments indicate that the 5' end of this cDNA clone is probably located only 13 nucleotides downstream of the actual cap site of the 56-kDa-protein mRNA. It consists of a 64-nucleotide-long 5'-non-coding segment, a coding segment of 1434 nucleotides terminated by a TAG triplet and a 141-nucleotide 3'-non-coding segment. The encoded protein of 478 amino acid residues has a molecular mass of 55335 Da and a single potential site for N-glycosylation. The protein contains an excess of basic amino acids and most of them are localized in the carboxy-terminal half of the molecule. A single [35S]methionine-labeled 56-kDa protein was obtained using an SP64 construction to allow the cell-free transcription and translation of the cloned cDNA. Microinjection of this labeled protein in Xenopus oocytes indicates that the 56-kDa protein is cytoplasmic.

The IFI-56K and IFI-54K genes are transcriptionally stimulated when cells are treated by interferon. We have previously shown that the IFI-56K gene is in addition directly induced by poly(rI).poly(rC), and inducer of interferon-beta. Since the regulation of the IFI-56K and IFI-54K genes by interferon are very much alike, we tested whether the IFI-54K gene is also directly regulated by poly(rI).poly(rC). Treatment of various cell lines with poly(rI).poly(rC) leads to a clear accumulation of the IFI-54K mRNA to a level which sometimes even exceeds that obtained with high doses of interferon. Several interferon-resistant cell lines were investigated for the inducibility of both the IFI-56K and IFI-54K genes by interferons, poly(rI).poly(rC) and viruses (which are the natural inducers of interferon-alpha and -beta). Both genes appear to be coordinately regulated by these inducers. It was thus interesting to search for common regulatory element(s) in the control region of these two genes. The IFI-54K gene promoter region was isolated, from which a 520-base-pair segment was sequenced and compared with the promoter region of the IFI-56K gene that we had previously sequenced. The only homology was found is a well conserved 19-bp segment located just upstream of the TATA box of these genes; interestingly, this sequence is also homologous to the minimal region needed for the inducibility by poly(rI).poly(rC) of the interferon-beta gene. This conserved sequence might be responsible for the coordinate induction of the IFI-56K and IFI-54K genes by interferon, poly(rI).poly(rC) and viruses.

In order to investigate the molecular basis of the regulation of interferon-inducible genes, we isolated the promoter region of two such genes coding for the (2'-5')oligo(adenylate) synthetase and a 56-kDa protein (IFI-56K). The regions surrounding the cap site were sequenced and compared with the sequences of vertebrate and viral DNA present in the Genbank data bank. Small DNA segments were found in both genes which are homologous to part of the promoter region of other genes, such as those of interferon-beta, tumor necrosis factor beta, interleukin-2 and its receptor. Since these homologies were found located in functionally important regions of these genes, we tested whether their inducers also enhance the (2'-5')oligo(adenylate) synthetase and IFI-56K gene expression. We found that poly(rI).poly(rC) and interleukin-1, activators of the interferon-beta gene and of T lymphocytes respectively, are both able to enhance IFI-56K mRNA accumulation in all cell lines tested. Cycloheximide even superinduces this gene when added together with poly(rI).poly(rC) and interleukin-1 (but not when added with interferon). We showed that these inductions are direct and not mediated by interferon produced by cells in response to poly(rI).poly(rC) or interleukin-1. The promoter sequence analyses have thus led to the discovery of unexpected inducers, i.e. an interferon inducer such as poly(rI).poly(rC) is also able to directly induce a gene that is under the control of interferon.

The expression of the gene coding for the 26-kDa protein coinduced with human beta-interferon (HuIFN-beta) in human fibroblasts has been measured by cytoplasmic dot hybridization in WISH cells. The production of the 26-kDa-protein mRNA is not induced by poly(I).poly(C) but maximally induced by cycloheximide alone. In contrast, HuIFN-beta is induced by poly(I).poly(C) and not by cycloheximide. WISH cells showed in addition a low constitutive level of 26-kDa-protein mRNA prior to induction. These results were confirmed by sizing the RNAs by Northern blot analysis. Pretreatment with partially purified or pure IFN-beta has only a slight effect on 26-kDa protein mRNA production. We have also determined the kinetics of induction and the amount of inducer required for an optimal induction of the 26-kDa-protein mRNA in WISH cells. This mRNA was thus maximally induced in WISH cells in the absence of detectable IFN-beta; it represents about 0.05% of poly(A)-rich mRNA in cycloheximide-induced WISH cells. We had already found that the 26-kDa-protein does not share the general characteristics of interferons. These results suggest that HuIFN-beta and the 26-kDa-protein genes are differently regulated.

Synthesis of polypeptide chains coded by exogenous messenger RNAs is inhibited in cell-free extracts from interferon-treated mouse L cells, due to a "deficiency" in some specific tRNA species. A detailed analysis shows that polypeptide chain elongation is blocked and incomplete chains are formed. After a few minutes, however, initiation of new polypeptide chains is also blocked. Messenger RNA still binds to ribosomes but initiator Met-tRNA(FMET) binding is inhibited. The block in initiation appears to be secondary to the block in elongation.