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Interleukin (IL)-33 is crucially involved in liver pathology and drives hepatoprotective functions. However, the regulation of IL-33 by cytokines of the IL-6 family, including oncostatin M (OSM) and IL-6, is not well studied. The aim of the present study was to determine whether OSM mediates regulation of IL-33 expression in liver cells. Intramuscular administration in mice of an adenovirus encoding OSM (AdOSM) leads to increase in expression of OSM in muscles, liver, and serum of AdOSM-infected mice compared with control mice. The increase of circulating OSM markedly regulated mRNA of genes associated with blood vessel biology, chemotaxis, cellular death, induction of cell adhesion molecules, and the alarmin cytokine IL-33 in liver. Steady-state IL-33 mRNA was upregulated by OSM at an early phase (8 h) following AdOSM infection. At the protein level, the expression of IL-33 was significantly induced in liver endothelial cells [liver sinusoidal endothelial cells (LSEC) and vascular endothelial cells] with a peak at 8 days post-AdOSM infection in mice. In addition, we found OSM-stimulated human microvascular endothelial HMEC-1 cells and human LSEC/TRP3 cells showed a significant increase in expression of IL-33 mRNA in a dose-dependent manner in cell culture. The OSM-mediated overexpression of IL-33 was associated with the activation/enrichment of CD4(+)ST2(+) cells in liver of AdOSM-infected mice compared with adenovirus encoding green fluorescent protein-treated control mice. In summary, these data suggest that the cytokine OSM regulates the IL-33 expression in liver endothelial cells in vivo and in HMEC-1/TRP3 cells in vitro and may specifically expand the target CD4(+)ST2(+) cells in liver., (Copyright © 2015 the American Physiological Society.)

Background & Aims: Infection with hepatitis C virus is a worldwide health problem. An inadequate Th2 cytokine response promotes the fibrosis-cirrhosis fate. Immune-modulating molecules favoring a Th2 profile, such as HLA-G molecules of the HLA class Ib family, may play a role in chronic hepatitis. HLA-G contributes to the escape of tumors, and their involvement in viral infections has been increasingly described. The aim of this work was to study the expression of HLA-G in the liver, its cellular source and its regulation in cases of chronic C hepatitis., Methods: HLA-G cells in blocks of liver derived from patients infected with HCV were labeled by immunohistochemistry and enumerated. Double immunofluorescence allowed the identification of the cellular source. HLA-G secretion by a human mast cell line was quantified by ELISA after various stimulations. After treatment with IFN-α, real-time PCR was performed to determine the kinetics of cytokine expression profiles, followed by heat map clustering analysis., Results: The number of HLA-G+ cells was significantly associated with the area of fibrosis. For the first time, we identify the HLA-G+ cells as being mast cells. HLA-G secretion was significantly induced in human mast cells stimulated by IL-10 or interferons of class I. The transcriptome of the secretome of this cell line stimulated by IFN-α revealed that (i) the HLA-G gene is upregulated late, and that (ii) T lymphocytes and NK cells are recruited., Conclusions: These findings suggest an autocrine loop in the genesis of HCV liver fibrosis, based on mast cells expressing HLA-G., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Bone resorption by osteoclasts and bone formation by osteoblasts are tightly coupled processes implicating factors in TNF, bone morphogenetic protein, and Wnt families. In osteoimmunology, macrophages were described as another critical cell population regulating bone formation by osteoblasts but the coupling factors were not identified. Using a high-throughput approach, we identified here Oncostatin M (OSM), a cytokine of the IL-6 family, as a major coupling factor produced by activated circulating CD14+ or bone marrow CD11b+ monocytes/macrophages that induce osteoblast differentiation and matrix mineralization from human mesenchymal stem cells while inhibiting adipogenesis. Upon activation of toll-like receptors (TLRs) by lipopolysaccharide or endogenous ligands, OSM was produced in classically activated inflammatory M1 and not M2 macrophages, through a cyclooxygenase-2 and prostaglandin-E2 regulatory loop. Stimulation of osteogenesis by activated monocytes/macrophages was prevented using neutralizing antibodies or siRNA to OSM, OSM receptor subunits gp130 and OSMR, or to the downstream transcription factor STAT3. The induced osteoblast differentiation program culminated with enhanced expression of CCAAT-enhancer-binding protein δ, Cbfa1, and alkaline phosphatase. Overexpression of OSM in the tibia of mice has led to new bone apposition with no sign of bone resorption. Two other cytokines have also a potent role in bone formation induced by monocytes/macrophages and activation of TLRs: IL-6 and leukemia inhibitory factor. We propose that during bone inflammation, infection, or injury, the IL-6 family signaling network activated by macrophages and TLR ligands stimulates bone formation that is largely uncoupled from bone resorption and is thus an important target for anabolic bone therapies., (Copyright © 2012 AlphaMed Press.)

The development of inflammatory granulomas around infected Kupffer cells is necessary for hepatic parasite clearance during visceral leishmaniasis. Invariant NKT (iNKT) cells are predominant T cells in the mouse liver and can synthesize large quantities of IL-4 and IFN-γ, two cytokines involved in granuloma formation. This study analyzed the role of iNKT cells in the hepatic immune response during Leishmania donovani infection, using a murine model of wild-type (WT) and iNKT cell-deficient (Jα18⁻/⁻) C57BL/6 mice sacrificed 15, 30 or 60 days post-infection. We recorded hepatic parasite loads, cytokine expression, and analyzed granulomatous response by immunohistochemistry and hepatic immune cell infiltration by flow cytometry. Whereas WT animals rapidly controlled the infection and developed an inflammatory response associated with a massive influx of iNKT cells observed by flow cytometry, Jα18⁻/⁻ mice had significantly higher parasitic loads on all time points. This lack of control of parasite burden was associated with a delay in granuloma maturation (28.1% of large granulomas at day 60 versus 50.7% in WT). Cytokine transcriptome analysis showed that mRNA of 90/101 genes encoding chemokines, cytokines and their receptors, was underexpressed in Jα18⁻/⁻ mice. Detection of IL-4 and TNF-α by ELISA in liver extracts was also significantly lower in Jα18⁻/⁻ mice. Consistent with flow cytometry analysis, cytokinome profile in WT mice showed a bias of expression towards T cell-chemoattractant chemokines on D15, and displayed a switch towards expression of granulocytes and/or monocytes -chemoattractant chemokines on D60. In Jα18⁻/⁻ mice, the significantly lower expression of CXCL5, MIP-2 and CCL2 mRNA was correlated with a defect in myeloperoxidase positive-cell attraction observed by immunohistochemistry and with a lower granulocyte and monocyte infiltration in the liver, as shown by flow cytometry. These data indicate that iNKT cells play a role in early and sustained pro-inflammatory cytokine response warranting efficient organization of hepatic granulomas and parasite clearance.

IL-27, consisting of the subunits IL-27p28 and Epstein-Barr virus-induced gene 3 (EBI3), is a heterodimeric cytokine belonging to the IL-6/IL-12 family of cytokines. IL-27p28 is a four-helical cytokine requiring association with the soluble receptor EBI3 to be efficiently secreted and functionally active. Computational and biological analyses of the IL-27 binding site 1 to its receptor revealed important structural proximities with the ciliary neurotrophic factor group of cytokines and highlighted the contribution of p28 Trp(97), as well as of EBI3 Phe(97), Asp(210), and Glu(159), as key residues in the interactions between both cytokine subunits. WSX-1 (IL-27R) and gp130 compose the IL-27 receptor-signaling complex, recruiting the STAT-1 and STAT-3 pathways. A study of IL-27 binding site 3 showed that Trp(197) was crucial for the cytokine's interaction with gp130, but that the mutated cytokine still recognized IL-27R on the cell surface. IL-27 exerts both pro- and anti-inflammatory functions, promoting proliferation and differentiation of Th1 and inhibiting Th17 differentiation. Our results led us to develop mutated forms of human and mouse IL-27 with antagonistic activities. Using an in vivo mouse model of concanavalin A-induced Th1-cell-mediated hepatitis, we showed that the murine IL-27 antagonist W195A decreased liver inflammation by downregulating the synthesis of CXCR3 ligands and several acute phase proteins. Together, these data suggest that IL-27 antagonism could be of interest in down-modulating acute IL-27-driven Th1-cell-mediated immune response.

Interleukin-33 (IL-33), the most recently identified member of the IL-1 family, induces synthesis of T Helper 2 (Th2)-type cytokines via its heterodimeric ST2/IL-1RAcP receptor. Th2-type cytokines play an important role in fibrosis; thus, we investigated the role of IL-33 in liver fibrosis. IL-33, ST2 and IL-1RAcP gene expression was analysed in mouse and human normal (n= 6) and fibrotic livers (n= 28), and in human hepatocellular carcinoma (HCC; n= 22), using real-time PCR. IL-33 protein was detected in normal and fibrotic liver sections and in isolated liver cells using Western blotting and immunolocalization approaches. Our results showed that IL-33 and ST2 mRNA was overproduced in mouse and human fibrotic livers, but not in human HCC. IL-33 expression correlated with ST2 expression and also with collagen expression in fibrotic livers. The major sources of IL-33 in normal liver from both mice and human beings are the liver sinusoidal endothelial cells and, in fibrotic liver, the activated hepatic stellate cells (HSC). Moreover, IL-33 expression was increased in cultured HSC when stimulated by pro-inflammatory cytokines. In conclusion, IL-33 is strongly associated with fibrosis in chronic liver injury and activated HSC are a source of IL-33.

Interleukin-31 (IL-31) is a recently described T cell-derived cytokine, mainly produced by T helper type 2 cells and related to the IL-6 cytokine family according to its structure and receptor. IL-31 is the ligand for a heterodimeric receptor composed of a gp130-like receptor (GPL) associated with the oncostatin M receptor (OSMR). A link between IL-31 and atopic dermatitis was shown by studying the phenotype of IL-31 transgenic mice and IL-31 gene haplotypes in patients suffering from dermatitis. In this study, we generated a potent IL-31 antagonist formed by external portions of OSMR and GPL fused with a linker. This fusion protein, OSMR-L-GPL, consisting of 720 amino acids, counteracted the binding of IL-31 to its membrane receptor complex and the subsequent signaling events involving the STATs and MAPK pathways. Neutralizing effects were found in IL-31-sensitive cell lines, including brain-derived cells and primary cultures of keratinocytes.

Interleukin (IL)-31 is a recently described cytokine, preferentially produced by T helper 2 lymphocytes and associated with skin diseases, such as atopic dermatitis. IL-31 is a member of the four alpha-helix bundle cytokine family and is related to the IL-6 subgroup. Its heterodimeric membrane receptor is composed of the gp130-like receptor (GPL) subunit associated to the oncostatin M receptor subunit. We identified critical amino acids implicated in the ligand receptor interaction by computational analysis combined with site-directed mutagenesis. Six IL-31 residues selected for their putative involvement in cytokine receptor contact sites were alanine-substituted, and the corresponding proteins were expressed in mammalian and bacterial systems. Biochemical, membrane binding, cell signaling, and cell proliferation analyses showed that mutation E44A, E106A, or H110A abolished IL-31 binding to GPL and the subsequent signaling events. A second ligand receptor-binding site involved Lys(134), with alanine substitution leading to a protein that still binds GPL, but is unable to recruit the second receptor subunit and the subsequent signaling pathways. The results indicate that IL-31 recognizes its receptor complex through two different binding sites, and we propose a three-dimensional model for IL-31.

Prokineticin 1 and 2 (PROK1 and PROK2) are two small proteins largely expressed in inflammatory tissues and involved in monocyte activation and differentiation. The focus of this study was to evaluate whether PROK1 was able to induce chemokine secretion in human monocytes, in monocyte-derived macrophages and in monocyte-derived dendritic cells, an aspect not addressed thus far. Here, we show for the first time, using flow cytometry, that PROK receptors 1 and 2 are present on the surface of human monocytes. Subsequently, monocytes were selected to investigate the chemokine response after stimulation by PROK1. Our results show that only three chemokines (CCL4, CXCL1 and CXCL8) were significantly induced at both the transcript and protein level, and that PROK1 induces most potently CXCL8, in a dose-dependent manner. From a mechanistic point of view, by blocking independently Galphai protein or intracellular calcium, monocytes lose the ability to secrete CXCL8 in response to PROK1. Finally, we observed that CCL4, CXCL1 and CXCL8 secretion, following PROK1 induction, is only observed in monocytes and not in monocyte-derived macrophages and dendritic cells. Our results demonstrate that, in vitro, the differentiation status of monocytes influences chemokine production after stimulation by PROK1, and that this chemokine production is geared toward a pro-inflammatory response. This could represent a novel amplification loop of leukocyte recruitment, extravasation and tissue invasion.

Ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin are members of the four-helix bundle cytokine family. These proteins signal through a common tripartite receptor composed of leukemia inhibitory factor receptor, gp130, and ciliary neurotrophic factor receptor alpha. Binding to ciliary neurotrophic factor receptor alpha occurs through an interaction site located at the C terminus of the cytokine AB loop and alphaD helix, known as site 1. In the present study, we have generated a model of neuropoietin and identified a conserved binding site for the three cytokines interacting with ciliary neurotrophic factor receptor alpha. To identify the counterpart of this site on ciliary neurotrophic factor receptor alpha, its cytokine binding domain was modeled, and the physicochemical properties of its surface were analyzed. This analysis revealed an area displaying properties complementary to the site 1 of ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin. Based on our computational predictions, residues were selected for their potential involvement in the ciliary neurotrophic factor receptor alpha binding epitope, and site-directed mutagenesis was carried out. Biochemical, cell proliferation, and cell signaling analyses showed that Phe(172) and Glu(286) of ciliary neurotrophic factor receptor alpha are key interaction residues. Our results demonstrated that ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin share a conserved binding site on ciliary neurotrophic factor receptor alpha.

Chronic inflammatory diseases are characterized by local tissue injury caused by immunocompetent cells, in particular CD4(+) T lymphocytes, that are involved in the pathogenesis of these disorders via the production of distinctive sets of cytokines. Here, we have characterized single CD4(+) T cells that infiltrate inflamed tissue taken from patients with psoriasis, Crohn's disease, rheumatoid arthritis, or allergic asthma. Results from a cytokine production and gene profile analysis identified a population of in vivo differentiatedretinoid-related orphan receptor gamma-expressing T cells, producing high levels of IL-17, that can represent up to 30% of infiltrating T lymphocytes. Activated Th17 cells produced IL-26, TNF-alpha, lymphotoxin-beta, and IL-22. IL-17 and IL-22 concentrations secreted by tissue infiltrating Th17 cells could reach up to 100 nM and were inversely correlated with the production of Th1- and Th2-associated cytokines. In addition, tissue-infiltrating Th17 cells are also characterized by high cell surface expression of CCR6, a chemokine receptor that was not expressed by Th1 and Th2 cells, isolated from the same lesions, and by the production of CCL20/MIP3alpha, a CCR6 ligand, associated with tissue infiltration. Culture supernatants of activated Th17 cells, isolated from psoriatic lesions, induced the expression of gene products associated with inflammation and abnormal keratinocyte differentiation in an IL-17 and IL-22-dependent manner. These results show that tissue-infiltrating Th17 cells contribute to human chronic inflammatory disease via the production of several inflammatory cytokines and the creation of an environment contributing to their migration and sequestration at sites of inflammation.

Some observations have suggested that cells from the central nervous system (CNS) could present exogenous antigens on major histocompatibility complex (MHC) class I molecules to CD8(+) T cells (a process called cross-presentation). Microglia are the major myeloid immunocompetent cells of the CNS. When activated, following the injury of the nervous parenchyma, they become fully competent antigen-presenting cells (APC) that prime CD4(+) T lymphocytes. We therefore tested the cross-presentation capacity of murine microglia. We report that a microglial cell line (C8-B4), neonatal microglia, and interestingly adult microglia cross-present soluble exogenous antigen (ovalbumin) to a OVA-specific CD8(+) T-cell hybridoma and cross-prime OVA-specific naive OT-1 CD8(+) T cells. In both these cases, C8-B4 and neonatal microglia cross-present OVA as well as peritoneal macrophages. Although cross-presentation by adult microglia is less efficient, it is increased by GM-CSF and CpG oligodeoxynucleotide (ODN) stimulation. Using microglial cells either exposed to an inhibitor of proteasome, lactacystin, or purified from TAP(-/-) mice, we demonstrate that the microglia cross-present antigen in proteasome- and TAP-dependant pathways, respectively. Last, microglia purified from adult mice injected intracerebrally with OVA efficiently stimulate OVA-specific CD8(+) T cells, thereby showing that microglia take up and process exogenous antigen into MHC class I in vivo. This first demonstration of the cross-presentation property of microglia offers novel therapeutic approaches to modulate CD8 T-cell responses in the brain., ((c) 2007 Wiley-Liss, Inc.)

Tumor-associated macrophages (TAMs), the most abundant immunosuppressive cells in the tumor microenvironment, originate from blood monocytes and exhibit an IL-10(high)IL-12(low) M2 profile. The factors involved in TAM generation remain unidentified. We identify here leukemia inhibitory factor (LIF) and IL-6 as tumor microenvironmental factors that can promote TAM generation. Ovarian cancer ascites switched monocyte differentiation into TAM-like cells that exhibit most ovarian TAM functional and phenotypic characteristics. Ovarian cancer ascites contained high concentrations of LIF and IL-6. Recombinant LIF and IL-6 skew monocyte differentiation into TAM-like cells by enabling monocytes to consume monocyte-colony-stimulating factor (M-CSF). Depletion of LIF, IL-6, and M-CSF in ovarian cancer ascites suppressed TAM-like cell induction. We extended these observations to different tumor-cell line supernatants. In addition to revealing a new tumor-escape mechanism associated with TAM generation via LIF and IL-6, these findings offer novel therapeutic perspectives to subvert TAM-induced immunosuppression and hence improve T-cell-based antitumor immunotherapy efficacy.

Neutrophils are professional phagocytes that migrate early, in high number, to the infection sites. Our study has analyzed how neutrophils cross-present antigens and influence CD8+ T-cell responses. By using highly purified neutrophils from peritoneal exudates and bone marrow, we have shown that neutrophils cross-present ovalbumin to a CD8+ T-cell hybridoma and to naive CD8+ T cells from OT1 transgenic mice. Cross-presentation by neutrophils was TAP and proteasome dependent and was as efficient as in macrophages. Moreover, it actually occurred earlier than in professional antigen-presenting cells. Peritoneal exudate neutrophils from mice injected intraperitoneally with ovalbumin also cross-presented ovalbumin, proving that neutrophils take up and present exogenous antigens into major histocompatibility complex I (MHC I) molecules in vivo. We then evaluated the in vivo influence of antigen cross-presentation by neutrophils on CD8+ T-cell response using beta2-microglobulin-deficient mice transferred with OT1 CD8+ T cells and injected with ovalbumin-pulsed neutrophils. Four days after neutrophil injection, OT1 cells proliferated and expressed effector functions (IFN-gamma production and cytolysis). They also responded efficiently to a rechallenge with ovalbumin-pulsed dendritic cells in CFA. These data are the first demonstration that neutrophils cross-prime CD8+ T cells in vivo and suggest that they may constitute, together with professional antigen-presenting cells, an attractive target to induce cytotoxic T cells in vaccines.

The long pentraxin (PTX) 3 is produced by macrophages and myeloid dendritic cells in response to Toll-like receptor agonists and represents a nonredundant component of humoral innate immunity against selected pathogens. We report that, unexpectedly, PTX3 is stored in specific granules and undergoes release in response to microbial recognition and inflammatory signals. Released PTX3 can partially localize in neutrophil extracellular traps formed by extruded DNA. Eosinophils and basophils do not contain preformed PTX3. PTX3-deficient neutrophils have defective microbial recognition and phagocytosis, and PTX3 is nonredundant for neutrophil-mediated resistance against Aspergillus fumigatus. Thus, neutrophils serve as a reservoir, ready for rapid release, of the long PTX3, a key component of humoral innate immunity with opsonic activity.

Cutaneous inflammatory diseases such as psoriasis vulgaris and atopic dermatitis are associated with altered keratinocyte function, as well as with a particular cytokine production profile of skin-infiltrating T lymphocytes. In this study we show that normal human epidermal keratinocytes express a functional type II oncostatin-M (OSM) receptor (OSMR) consisting of the gp130 and OSMRbeta components, but not the type I OSMR. The type II OSMR is expressed in skin lesions from both psoriatic patients and those with atopic dermatitis. Its ligand, OSM, induces via the recruitment of the STAT3 and MAP kinase pathways a gene expression profile in primary keratinocytes and in a reconstituted epidermis that is characteristic of proinflammatory and innate immune responses. Moreover, OSM is a potent stimulator of keratinocyte migration in vitro and increases the thickness of a reconstituted epidermis. OSM transcripts are enhanced in both psoriatic and atopic dermatitic skin as compared with healthy skin and mirror the enhanced production of OSM by T cells isolated from diseased lesions. Results from a microarray analysis comparing the gene-modulating effects of OSM with those of 33 different cytokines indicate that OSM is a potent keratinocyte activator similar to TNF-alpha, IL-1, IL-17, and IL-22 and that it acts in synergy with the latter cytokines in the induction of S100A7 and beta-defensin 2 expression, characteristic of psoriatic skin. Taken together, these results demonstrate that OSM and its receptor play an important role in cutaneous inflammatory responses in general and that the specific effects of OSM are associated with distinct inflammatory diseases depending on the cytokine environment.

Activation of the signaling transduction pathways mediated by oncostatin M (OSM) requires the binding of the cytokine to either type I OSM receptor (leukemia inhibitory factor receptor/gp130) or to type II OSM receptor (OSMR/gp130). In the present work we have developed an enzyme-linked immunosorbent assay detecting a soluble form of OSMR (sOSMR) secreted by glioblastoma, hepatoma, and melanoma tumor cell lines. sOSMR was also present in sera of healthy individuals, with increased levels in multiple myeloma. Molecular cloning of a corresponding cDNA was carried out, and it encoded for a 70-kDa protein consisting of a half cytokine binding domain containing the canonical WSXWS motif, an immunoglobulin-like domain, and the first half of a second cytokine binding domain with cysteines in fixed positions. Analysis of the soluble receptor distribution revealed a preferential expression in lung, liver, pancreas, and placenta. sOSMR was able to bind OSM and interleukin-31 when associated to soluble gp130 or soluble interleukin-31R, respectively, and to neutralize both cytokine properties. We have also shown that OSM could positively regulate the synthesis of its own soluble receptor in tumor cells.

Ciliary neurotrophic factor (CNTF) receptor controls a pathway supporting the differentiation and survival of a wide range of neural cell types during development and in adulthood. Cardiotrophin-like cytokine (CLC)-cytokine-like factor 1 (CLF) composite cytokine is a second ligand for the CNTF alpha-component receptor (CNTFRalpha). This composite cytokine is built on the structural model of IL-12, with a complex formed by a four-helix bundle type I cytokine, CLC (also referred to as CLCF1), bound to a soluble receptor subunit, CLF (also known as CRLF1). We have reported mutations in the chaperone soluble receptor CLF, causing cold-induced sweating syndrome (CISS). In this study, we studied the CLC-mutated alleles in a patient suffering from a similar disease. This patient was compound heterozygous for two different CLC mutations. The first allele was inactivated by a stop codon at position 107 (Y107X). In the second allele, a R197L mutation in the CLC-predicted binding site to the CNTFRalpha was detected. Functional analysis of the mutated protein revealed an incapacity for R197L CLC to bind to CNTFRalpha and activate the subsequent signaling events. Structural and docking interaction studies showed that the R197L substitution destabilized the contact site between CLC and CNTFRalpha.

Sympathetic neurons are generated through a succession of differentiation steps that initially lead to noradrenergic neurons innervating different peripheral target tissues. Specific targets, like sweat glands in rodent footpads, induce a change from noradrenergic to cholinergic transmitter phenotype. Here, we show that cytokines acting through the gp 130 receptor are present in sweat glands. Selective elimination of the gp 130 receptor in sympathetic neurons prevents the acquisition of cholinergic and peptidergic features (VAChT, ChT1, VIP) without affecting other properties of sweat gland innervation. The vast majority of cholinergic neurons in the stellate ganglion, generated postnatally, are absent in gp 130-deficient mice. These results demonstrate an essential role of gp 130-signaling in the target-dependent specification of the cholinergic neurotransmitter phenotype.

TLRs are involved in innate cell activation by conserved structures expressed by microorganisms. Human T cells express the mRNA encoding most of TLRs. Therefore, we tested whether some TLR ligands may modulate the function of highly purified human CD4+ T lymphocytes. We report that, in the absence of APCs, flagellin (a TLR5 ligand) and R-848 (a TLR7/8 ligand) synergized with suboptimal concentrations of TCR-dependent (anti-CD3 mAb) or -independent stimuli (anti-CD2 mAbs or IL-2) to up-regulate proliferation and IFN-gamma, IL-8, and IL-10 but not IL-4 production by human CD4+ T cells. No effect of poly(I:C) and LPS, ligands for TLR3 and TLR4, respectively, was detected. We also observed that CD4+CD45RO+ memory T cell responses to TLR ligands were more potent than those observed with CD4+CD45RA+ naive T cells. Moreover, among the memory T cells, CCR7- effector cells were more sensitive to TLR ligands than CCR7+ central memory cells. These data demonstrate for the first time a direct effect of TLR5 and TLR7/8 ligands on human T cells, and highlight an innate arm in T cell functions. They also suggest that some components from invading microorganisms may directly stimulate effector memory T cells located in tissues by up-regulating cytokine and chemokine production.

Human interleukin-6 (hIL-6) is a pleiotropic mediator of activation and proliferation across a large number of different cell types. Human herpesvirus-8 (HHV-8) has been associated with classical and AIDS-related Kaposi's sarcoma (KS). HHV-8 encodes viral IL-6 (vIL-6), a functional homolog of human interleukin-6, that promotes the growth of KS and of some lymphoma cells. Signaling induced by human IL-6 requires recruitment of the glycoprotein gp130, which acts as the signal transducing chain, and of IL-6Ralpha, which is necessary for cognate recognition and high affinity receptor complex formation. In contrast, the formation of a functional complex between vIL-6 and gp130 does not require the presence of IL-6Ralpha. The physico-chemical properties of vIL-6 have been analyzed and compared to those of hIL-6 and of the receptor chains, gp130 and IL-6Ralpha. Interaction sites on vIL-6 involve more hydrophobic residues than those of hIL-6. The electrostatic fields induced by vIL-6 and IL-6Ralpha are repulsive and prevent interaction between vIL-6 and IL-6Ralpha, whereas the electrostatic field induced by hIL-6 steers the complex formation with IL-6Ralpha. Subsequently, electrostatic binding free energy in the vIL-6/IL-6Ralpha complex is destabilizing, whereas it is stabilizing in the complex comprising hIL-6. These properties result from charge reversals between viral and human IL-6, an unusual phenomenon of amino acid substitutions within a homologous protein family. This suggests a selection pressure for vIL-6 to by-pass the IL-6Ralpha control of host defense against virus infection. This selection pressure has yielded the reversal of electrostatic properties of vIL-6 when compared to hIL-6.

The recently identified IL-6 family member cardiotrophin-like cytokine (also named novel neurotrophin-1 or B cell stimulating factor-3) forms a secreted complex with cytokine-like factor-1 which binds and activates the tripartite ciliary neurotrophic factor receptor. The striking differences between the phenotype of mice in which either the ciliary neurotrophic factor or its receptor are inactivated suggest that the cardiotrophin-like cytokine/cytokine-like factor-1 complex could be the developmentally important ciliary neurotrophic factor receptor ligand. Cardiotrophin-like cytokine is also produced in the immune system and has been reported to activate B cells in vivo and in vitro. B cells do not express the ciliary neurotrophic factor receptor suggesting the existence of an alternative receptor. We produced the cardiotrophin-like cytokine/cytokine-like factor-1 complex tagged with a Bir A biotin ligase AviTag peptide substrate. This cytokine could be efficiently biotinylated in vitro with Bir A. It was subsequently validated as a sensitive tool for ciliary neurotrophic factor receptor detection by flow cytometry and for magnetic-activated cell sorting. It was also shown to allow the detection of a specific receptor by activated B cells. Whereas binding to cells expressing the ciliary neurotrophic factor receptor could be prevented by competition with ciliary neurotrophic factor, binding to B cells was not. The biotinylated cardiotrophin-like cytokine/cytokine-like factor-1 complex therefore represents a new reagent to study ciliary neurotrophic factor and cardiotrophin-like cytokine receptor expression and for the identification of the putative cardiotrophin-like cytokine B cell receptor. It further validates the use of biotin ligase catalysed biotinylation for the detection of cytokine receptors.

BACKGROUND: The ciliary neurotrophic factor (CNTF) receptor is composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor, associated with a non-signalling CNTF binding receptor alpha component (CNTFR). This tripartite receptor has been shown to play important roles in the development of motor neurons, but the identity of the relevant ligand(s) is still not clearly established. Recently, we have identified two new ligands for the CNTF receptor complex. These are heterodimeric cytokines composed of cardiotrophin-like cytokine (CLC) associated either with the soluble receptor subunit cytokine-like factor-1 (CLF) or the soluble form of the binding receptor itself (sCNTFR). RESULTS: Here we show that, during development, clc is expressed in lung, kidney, vibrissae, tooth, epithelia and muscles during the period of development corresponding to when motoneuron loss is observed in mice lacking a functional CNTF receptor. In addition, we demonstrate that it is co-expressed at the single cell level with clf and cntfr, supporting the idea that CLC might be co-secreted with either CLF or sCNTFR. CONCLUSION: This expression pattern is in favor of CLC, associated either with CLF or sCNTFR, being an important player in the signal triggered by the CNTF receptor being required for motoneuron development.

The cytokines of the interleukin-6 family are multifunctional proteins that regulate cell growth, differentiation, and other cell functions in a variety of biological systems including the immune, inflammatory, hematopoietic, and nervous systems. One member of this family, ciliary neurotrophic factor (CNTF), displays biological functions more restricted to the neuromuscular axis. We have recently identified two additional ligands for the CNTF receptor complex. Both are composite cytokines formed by cardiotrophin-like cytokine (CLC) associated to either the soluble type I cytokine receptor CLF or the soluble form of CNTF receptor alpha (CNTFRalpha). The present study was aimed at analyzing the interactions between the cytokine CLC and its different receptor chains. For this purpose, we modeled CLC/receptor interactions to define the residues potentially involved in the contact sites. We then performed site-directed mutagenesis on these residues and analyzed the biological interactions between mutants and receptor chains. Importantly, we found that CLC interacts with the soluble forms of CNTFRalpha and CLF via sites 1 and 3, respectively. For site 1, the most crucial residues involved in the interaction are Trp67, Arg170, and Asp174, which interact with CNTFRalpha. Surprisingly, the residues that are important for the interaction of CLC with CLF are part of the conserved FXXK motif of site 3 known to be the interaction site of LIFRbeta. Obtained results show that the Phe151 and Lys154 residues are effectively involved in the interaction of CLC with LIFRbeta. This study establishes the molecular details of the interaction of CLC with CLF, CNTFRalpha, and LIFRbeta and helps to define the precise role of each protein in this functional receptor complex.

Gp130-like receptor (GPL) is a newly identified cytokine receptor. A recent study reported the involvement of GPL, together with OSMR, in the formation of the receptor complex for IL-31, a novel immune cytokine with a skin tropism. In the present work, we analyzed the signaling properties of IL-31 in glioblastoma and melanoma tumor cells. We demonstrate that in response to IL-31, its receptor complex recruits Jak1, Jak2, STAT1, -3, -5 signaling pathways, as well as the Pi3 kinase / AKT cascade. SHP-2 and Shc adapter molecules are also recruited and contribute to an increased activation of the MAP kinase pathway in response to IL-31. Different responses were observed depending on the expression of short or long GPL receptor isoform within the studied cell lines. We show that the short form of GPL receptor exerts a profound inhibitory effect on the signaling of IL-31 and behaves as a dominant negative receptor.

Ciliary neurotrophic factor (CNTF) is a neuroprotective cytokine initially identified in chick embryo. It has been evaluated for the treatment of neurodegenerative diseases. CNTF also acts on non-neuronal cells such as oligodendrocytes, astrocytes, adipocytes and skeletal muscles cells. CNTF has regulatory effects on body weight and is currently in clinical trial for the treatment of diabetes and obesity. CNTF mediates its function by activating a tripartite receptor comprising the CNTF receptor alpha chain (CNTFRalpha), the leukemia inhibitory factor receptor beta chain (LIFRbeta) and gp130. Human, rat and chicken CNTF have been expressed as recombinant proteins, and most preclinical studies in murine models have been performed using rat recombinant protein. Rat and human CNTF differ in their fine specificities: in addition to CNTFR, rat CNTF has been shown to activate the LIFR (a heterodimer of LIFRbeta and gp130), whereas human CNTF can bind and activate a tripartite receptor comprising the IL-6 receptor alpha chain (IL-6Ralpha) and LIFR. To generate tools designed for mouse models of human diseases; we cloned and expressed in E. coli both mouse CNTF and the CNTFRalpha chain. Recombinant mouse CNTF was active and showed a high level of specificity for mouse CNTFR. It shares the arginine residue with rat CNTF which prevents binding to IL-6Ralpha. It did not activate the LIFR at all concentrations tested. Recombinant mouse CNTF is therefore specific for CNTFR and as such represents a useful tool with which to study CNTF in mouse models. It appears well suited for the comparative evaluation of CNTF and the two additional recently discovered CNTFR ligands, cardiotrophin-like cytokine\cytokine-like factor-1 and neuropoietin.

A structural profile-based computational screen was used to identify neuropoietin (NP), a new cytokine. The np gene is localized in tandem with the cardiotrophin-1 gene on mouse chromosome 7. NP shares structural and functional features with ciliary neurotrophic factor (CNTF), cardiotrophin-1, and cardiotrophin-like cytokine. It acts through a membrane receptor complex comprising CNTF receptor-alpha component (CNTFRalpha), gp130, and leukemia inhibitory factor receptor to activate signal transducer and activator of transcription 3 signaling pathway. NP is highly expressed in embryonic neuroepithelia. Strikingly, CNTFRalpha, but not its alternate ligands, CNTF and cardiotrophin-like cytokine, is expressed at the same developmental stages. NP is also observed in retina and to a lesser extent in skeletal muscle. Moreover, NP could sustain the in vitro survival of embryonic motor neurons and could increase the proliferation of neural precursors when associated to epidermal growth factor and fibroblast growth factor 2. Thus, NP is a new ligand for CNTFRalpha, with important implications for murine nervous system development.

We describe a novel cytokine receptor named GP130 Like receptor, or GPL, that displays similarities with the interleukin-6 and interleukin-12 family of signaling receptors. Four different isoforms diverging in their carboxyl terminus were isolated, corresponding to proteins encompassing 560, 610, 626, and 745 amino acids. Sequences included a signal peptide of 32 amino acids, followed by a cytokine binding domain containing four conserved cysteines, a WSDWS motif, and a region consisting of three fibronectin type III domain repeats. No immunoglobulin-like module was identified in the GPL sequences. The intracellular part of longer isoforms contained a proline-rich region defining a box1 motif for interaction with the Janus kinases. The Gpl gene is organized in 15 exons and is located on 5q11.2 in tandem with the gp130 gene. Both genes were only separated by 24 kilobases, with opposite transcriptional orientations. The GPL receptor displayed a 28% identity with gp130. Specific GPL transcripts were observed in tissues involved in reproduction. Transcripts were also found in blood cells and in bone marrow, revealing expression of GPL in all of the myelomonocytic lineage, from hematopoietic stem cells to activated dendritic cells. In monocytes and dendritic cells, expression of GPL was strongly up-regulated by interferon-gamma, indicating a possible involvement of GPL in Th1-type immune responses. The molecular basis of cell signaling mediated by GPL was studied using chimeric receptors where external portions of alpha or beta interleukin-5 receptor subunits were fused to the internal portion of GPL or of related receptors. Results indicated that association of GPL to the intracellular portions of gp130, or LIF receptor, allowed the signaling cascade.

The extracellular protein Spätzle is required for activation of the Toll signaling pathway in the embryonic development and innate immune defense of Drosophila. Spätzle is synthesized as a pro-protein and is processed to a functional form by a serine protease. We show here that the mature form of Spätzle triggers a Toll-dependent immune response after injection into the hemolymph of flies. Spätzle specifically bound to Drosophila cells and to Cos-7 cells expressing Toll. Furthermore, in vitro experiments showed that the mature form of Spätzle bound to the Toll ectodomain with high affinity and with a stoichiometry of one Spätzle dimer to two receptors. The Spätzle pro-protein was inactive in all these assays, indicating that the pro-domain sequence, which is natively unstructured, acts to prevent interaction of the cytokine and its receptor Toll. These results show that, in contrast to the human Toll-like receptors, Drosophila Toll requires only an endogenous protein ligand for activation and signaling.

Leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and oncostatin M (OSM) are four helix bundle cytokines acting through a common heterodimeric receptor composed of gp130 and LIF receptor (LIFR). Binding to LIFR occurs through a binding site characterized by an FXXK motif located at the N terminus of helix D (site III). The immunoglobulin (Ig)-like domain of LIFR was modeled, and the physico-chemical properties of its Connolly surface were analyzed. This analysis revealed an area displaying properties complementary to those of the LIF site III. Two residues of the Ig-like domain of LIFR, Asp214 and Phe284, formed a mirror image of the FXXK motif. Engineered LIFR mutants in which either or both of these two residues were mutated to alanine were transfected in Ba/F3 cells already containing gp130. The F284A mutation impaired the biological response induced by LIF and CT-1, whereas the response to OSM remained unchanged. The Asp214 mutation did not alter the functional responses. The D214A/F284A double mutation, however, totally impaired cellular proliferation to LIF and CT-1 and partially impaired OSM-induced proliferation with a 20-fold increase in EC50. These results were corroborated by the analysis of STAT3 phosphorylation and Scatchard analysis of cytokine binding to Ba/F3 cells. Molecular modeling of the complex of LIF with the Ig-like domain of LIFR provides a clue for the superadditivity of the D214A/F284A double mutation. Our results indicate that LIF, CT-1, and OSM share an overlapping binding site located in the Ig-like domain of LIFR. The different behaviors of LIF and CT-1, on one side, and of OSM, on the other side, can be related to the different affinity of their site III for LIFR.

Background: The P-glycoprotein (P-gp), an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures., Results: Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression. The only effective proteins were IFNgamma and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF). As well as P-gp expression, the IL-6 type cytokines (but not IFNgamma) stimulated the expression of endogenous CNTF in astrocytes. In order to see whether an increased intracellular level of CNTF was necessary for induction of P-gp overexpression by IL-6 type cytokines, by the same cytokines analysis was carried out on astrocytes obtained from CNTF knockout mice. In these conditions, IFNgamma produced increased P-gp expression, but no overexpression of P-gp was observed with either IL-6, LIF or CT-1, pointing to a role of CNTF in the intracellular signalling pathway leading to P-gp overexpression. In agreement with this suggestion, application of exogenous CNTF (which is internalised with its receptor) produced an overexpression of P-gp in CNTF-deficient astrocytes., Conclusion: These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF. This regulation of P-gp may be one of the long searched for physiological roles of CNTF.

Interleukin (IL)-6-type cytokines are multifunctional proteins involved in cardiac hypertrophy and myocardial protection. Recent studies, performed on animal models, report the production of these cytokines by heart. The aim of this study was to analyse the capacity of myocytes and fibroblasts isolated from human atrium to secrete IL-6, leukaemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), IL-11, oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and the soluble receptor subunits sIL-6R and sgp130 during primary culture. We detected LIF, IL-11, sgp130 and a large amount of IL-6, but not OSM, CT-1, CNTF nor IL-6R in these culture supernatants. Both cardiomyocytes and fibroblasts are able to spontaneously produce IL-6. The increase of IL-6 production all along the culture period appears to be the consequence of fibroblast proliferation and gp130 stimulation. This is the first demonstration that human cardiac cells are able to secrete IL-6, but also LIF and IL-11 in vitro. These cytokines could be involved in an autocrine and/or a paracrine networks regulating myocardial cyto-protection, hypertrophy and fibrosis.

The heterodimeric cytokine composed of the soluble ciliary neurotrophic factor receptor (sCNTFR) and the IL-6 family member cardiotrophin-like cytokine (CLC) was recently identified as a new ligand for gp130-leukemia inhibitory factor receptor (LIFR) complex [Plun-Favreau, H., Elson, G., Chabbert, M., Froger, J., deLapeyriere, O., Lelievre, E., Guillet, C., Hermann, J., Gauchat, J. F., Gascan, H. & Chevalier, S. (2001) EMBO J. 20, 1692-1703]. This heterodimer shows overlapping biological properties with LIF. Although CLC contains a putative signal peptide and therefore should enter into the classical secretory pathway, the protein has been shown to be retained within transfected mammalian cells, unless coexpressed with either sCNTFR or cytokine like factor (CLF) [Elson, G. C., Lelievre, E., Guillet, C., Chevalier, S., Plun-Favreau, H., Froger, J., Suard, I., de Coignac, A. B., Delneste, Y., Bonnefoy, J. Y., Gauchat, J. F. & Gascan, H. (2000) Nat. Neurosci. 3, 867-872]. In the present study, we demonstrate that a fusion protein comprising CLC covalently coupled through a glycine/serine linker to sCNTFR (CC-FP) is efficiently secreted from transfected mammalian cells. CC-FP shows enhanced activities in respect to the CLC/sCNTFR native complex, on a number of cells expressing gp130 and LIFR on their surface. In addition, CC-FP is able to compete with CNTF for cell binding, indicating that both cytokines share binding epitope(s) expressed by their receptor complex. Analysis of the downstream signaling events revealed the recruitment by CC-FP of the signal transducer and activator of transcription (STAT)-3, Akt and mitogen-activated protein (MAP) kinase pathways. The monomeric bioactive CLC/sCNTFR fusion protein is therefore a powerful tool to study the biological role of the recently described cytokine CLC.

The oncostatin M receptor (OSMR) is part of a heterodimeric receptor complex that mediates signal transduction of the pleiotropic cytokine OSM via a signaling pathway involving Janus kinases (Jaks) and transcription factors of the signal transducers and activators of transcription (STAT) family. Upon heterologous expression of the OSMR in several cell lines, we observed that its surface expression was significantly enhanced by coexpression of the Janus kinases Jak1, Jak2, and Tyk2 but not Jak3. Chimeric receptors consisting of the extracellular region of the interleukin-5 receptor beta chain and the transmembrane and intracellular part of the OSMR were similarly up-regulated on the plasma membrane when Jak1 was coexpressed. The overall expression level of these constructs did not change significantly, but Jak1 coexpression increased the amount of endoglycosidase H-resistant, fully processed OSMR chimeras. Using mutated receptor and Jak1 constructs, we were able to demonstrate that association of Jak1 with the membrane proximal region of the receptor, but not its kinase activity, is necessary for this effect. Moreover, deletion of the OSMR box1/2 region also resulted in an improved surface expression indicating that this region may contain a signal preventing efficient receptor surface expression in the absence of associated Jaks. Finally we demonstrate that in Jak1-deficient cells, the endogenous OSMR is significantly down-regulated, an effect that can be reversed by transient expression of Jak1 in these cells.

Signal transducer and activator of transcription 3 (STAT3) transcription factors are cytoplasmic proteins that induce gene activation in response to cytokine receptor stimulation. Following tyrosine phosphorylation, STAT3 proteins dimerize, translocate to the nucleus, and activate specific target genes. This transcriptional activation by STAT3 proteins has been shown to require the recruitment of coactivators such as CREB-binding protein (CBP)/p300. In the present study, we show that steroid receptor coactivator 1, NcoA/SRC1a, originally identified as a nuclear receptor coactivator, also functions as a coactivator of STAT3 proteins. In coimmunoprecipitations, NcoA/SRC1a was found to associate with STAT3 following IL-6 stimulation of HepG2 hepatoma cells. Pull-down experiments indicated that the N-terminal part of NcoA/SRC1a associates with the activation domain of STAT3. Overexpression of NcoA/SRC1a or its SRC1e isoform enhanced transcriptional activation by STAT3 proteins in transient transfection experiments. This ability of NcoA/SRC1a to enhance STAT3 activity is dependent upon the presence of the CBP-interacting domain, activation domain 1. Using chromatin immunoprecipitation assays, we found that STAT3, NcoA/SRC1a, and CBP/p300 are simultaneously recruited to the p21(waf1) promoter following interleukin-6 stimulation. Taken together, these data suggest that CBP/p300 and NcoA/SRC1a may function in a common pathway to regulate STAT3 transcriptional activity.

Ciliary neurotrophic factor (CNTF) is a cytokine supporting the differentiation and survival of a number of neural cell types. Its receptor complex consists of a ligand-binding component, CNTF receptor (CNTFR), associated with two signaling receptor components, gp130 and leukemia inhibitory factor receptor (LIFR). Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second developmentally important ligand. We recently demonstrated that cardiotrophin-like cytokine (CLC) associates with the soluble orphan receptor cytokine-like factor-1 (CLF) to form a heterodimeric cytokine that displayed activities only on cells expressing the tripartite CNTF receptor on their surface. In this present study we examined the membrane binding of the CLC/CLF composite cytokine and observed a preferential interaction of the cytokine with the CNTFR subunit. Signaling pathways recruited by the CLC/CLF complex in human neuroblastoma cell lines were also analyzed in detail. The results obtained showed an activation of Janus kinases (JAK1, JAK2, and TYK2) leading to a tyrosine phosphorylation of the gp130 and LIFR. The phosphorylated signaling receptors served in turn as docking proteins for signal transducing molecules such as STAT3 and SHP-2. In vitro analysis revealed that the gp130-LIFR pathway could also stimulate the phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathways. In contrast to that reported before for CNTF, soluble CNTFR failed to promote the action CLC/CLF, and an absolute requirement of the membrane form of CNTFR was required to generate a functional response to the composite cytokine. This study reinforces the functional similarity between CNTF and the CLC/CLF composite cytokine defining the second ligand for CNTFR.

STAT3 transcription factors are cytoplasmic proteins that induce gene activation in response to cytokine receptor stimulation. Following tyrosine phosphorylation, STAT3 proteins dimerize, translocate into the nucleus, and activate specific target genes. Activation is transient, and down-regulation of STAT3 signaling occurs within a few hours. In this study, we show that cyclin D1 inhibits STAT3 activation. In co-immunoprecipitation and pull-down assays, cyclin D1 was found to associate with the activation domain of STAT3 upon interleukin-6 stimulation. Overexpression of cyclin D1 inhibited transcriptional activation by STAT3 proteins. This effect was not shared by cyclin E, was independent of association with Cdk4, and was unaffected by inhibitors of Cdk4. Whereas cyclin D1 had no effect on the steady-state level of STAT3 proteins in the cytoplasm, it was found to reduce the STAT3 nuclear level in HepG2 cells. These results suggest a model by which cyclin D1 is part of a feedback network controlling the down-regulation of STAT3 activity and highlight a new activity for this cell cycle regulatory protein.

Ciliary neurotrophic factor (CNTF) is involved in the survival of a number of different neural cell types, including motor neurons. CNTF functional responses are mediated through a tripartite membrane receptor composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor (LIFR), associated with a non-signalling CNTF binding receptor alpha component (CNTFR). CNTFR-deficient mice show profound neuronal deficits at birth, leading to a lethal phenotype. In contrast, inactivation of the CNTF gene leads only to a slight muscle weakness, mainly during adulthood, suggesting that CNTFR binds to a second ligand that is important for development. Modelling studies of the interleukin-6 family member cardiotrophin-like cytokine (CLC) revealed structural similarities with CNTF, including the conservation of a site I domain involved in binding to CNTFR. Co-expression of CLC and CNTFR in mammalian cells generates a secreted composite cytokine, displaying activities on cells expressing the gp130-LIFR complex on their surface. Correspondingly, CLC-CNTFR activates gp130, LIFR and STAT3 signalling components, and enhances motor neuron survival. Together, these observations demonstrate that CNTFR induces the secretion of CLC, as well as mediating the functional responses of CLC.

Ciliary neurotrophic factor (CNTF) is a cytokine supporting the differentiation and survival of various cell types in the peripheral and central nervous systems. Its receptor complex consists of a non-signaling alpha chain, CNTFR, and two signaling beta chains, gp130 and the leukemia inhibitory factor receptor (LIFR). Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second, developmentally important ligand. We have identified this factor as a stable secreted complex of cardiotrophin-like cytokine (CLC) and the soluble receptor cytokine-like factor-1 (CLF). CLF expression was required for CLC secretion, and the complex acted only on cells expressing functional CNTF receptors. The CLF/CLC complex activated gp130, LIFR and signal transducer and activator of transcription 3 (STAT3) and supported motor neuron survival. Our results indicate that the CLF/CLC complex is a second ligand for CNTFR with potentially important implications in nervous system development.

Signal transducers and activators of transcription (STAT) factors are cytoplasmic proteins that induce gene activation in response to cytokine receptor stimulation. Following tyrosine phosphorylation, STAT proteins dimerize, translocate into the nucleus, and activate specific target genes. Activation is transient, and down-regulation of STAT signaling occurs within a few hours. In the present study, we show that the cyclin-dependent kinase inhibitor p21(WAF1/CIP1/SDI1) inhibits STAT3 transcriptional activation. Following leukemia inhibitory factor stimulation, p21(WAF1/CIP1/SDI1) was found to associate with STAT3 proteins in coimmunoprecipitation and pull down assays. In vivo, overexpression of p21(WAF1/CIP1/SDI1) reduced transcriptional activation by STAT3 proteins but did not modify DNA binding activity. Interestingly, pull down experiments showed that p21(WAF1/CIP1/SDI1) could interact with the CREB-binding coactivator protein, and inhibition of STAT3 activity by p21(WAF1/CIP1/SDI1) did not occur when CREB-binding protein was overexpressed. These results suggest a model by which p21(WAF1/CIP1/SDI1) functions as an inhibitor of STAT3 signaling and highlight a new activity for this cyclin-dependent kinase inhibitor.

Ciliary neurotrophic factor (CNTF) participates in the survival of motor neurons and reduces the denervation-induced atrophy of skeletal muscles. Experiments performed in rats show a decrease in peripheral CNTF synthesis during aging, associated with an overexpression of its alpha-binding receptor component by skeletal muscles. Measurement of sciatic nerve CNTF production and of the muscular performance developed by the animals revealed a strong correlation between the two studied parameters (r = 0.8; p < 0.0003). Furthermore, the twitch and tetanic tensions measured in the isolated soleus skeletal muscle in 24-month-old animals increased 2. 5-fold by continuous in vivo administration of CNTF. Analyses of the activation level of leukemia inhibitory factor receptor beta- and signal transducer and activator of transcription 3-signaling molecules in response to exogenous CNTF revealed an increased tyrosine phosphorylation positively correlated with the twitch tension developed by the soleus muscle of the animals.

This study investigated whether modifications in the contractile function of rat muscle after 3 weeks of hindlimb suspension followed by different recovery periods were associated with changes in ciliary neurotrophic factor (CNTF) receptor alpha. CNTF receptor alpha was stronger in control extensor digitorum longus (EDL) than soleus. CNTF receptor alpha was increased in unweighted as compared to control soleus muscle, while no significant changes occurred in EDL muscle. Furthermore, CNTF level was not significantly modified in adult sciatic nerve.

Cardiotrophin 1 (CT-1) is a recently described cytokine sharing many biological properties with those reported previously for leukaemia inhibitory factor (LIF). In the present study we show that CT-1 binds to the KB epidermoid cancer cell surface through a tripartite receptor complex which includes the gp130 signal transducing protein, LIF receptor beta (LIFR beta) and a third component displaying a molecular weight of 80 kDa. CT-1 activates gp130 and LIFR beta transducing components, as attested by analysing their tyrosine phosphorylation level. The activation process is relayed to the nucleus by the recruitment of the STAT3 transcription factor. Analysis of KB cell line culture supernatants after CT-1 treatment indicates that CT-1 stimulates the production of interleukin 6 (IL-6) in a time- and dose-dependent manner. This stimulation of IL-6 production by CT-1 is associated with an increase in intracellular levels of IL-6 mRNA. This study suggests that at least in some pathological situations CT-1 might represent an immunomodulator regulating cytokine-induced gene products.

Cardiotrophin-1 (CT-1) is a recently isolated cytokine belonging to the interleukin-6 cytokine family. In the present study, we show that CT-1 binds to hepatocyte-derived cell lines of rat and human origin with high (Kd = 600-800 pM) and low (Kd approximately 3-6 nM) binding affinities. Treatment of HepG2 cells with CT-1 resulted in the induction of tyrosine phosphorylation of both transducing receptor subunits, gp130 and LIF receptor, and this phosphorylation was completely inhibited by a neutralizing anti-gp130 mAb. Addition of CT-1 to HepG2 or H35 cell cultures induced a dose-dependent production of several acute phase proteins (haptoglobin, fibrinogen, alpha1-acid glycoprotein, alpha2-macroglobulin). Moreover, the use of a neutralizing mAb to gp130 in cultures of HepG2 cells grown in the presence of CT-1, inhibited the induction of acute phase protein secretion, indicating an absolute requirement of gp130 in the formation of a functional CT-1 receptor. Altogether, these results suggest that CT-1 could play an important role in the regulation of hepatocyte metabolism in inflammatory responses.

Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) share common components in their multimeric receptors. Both cytokine receptors contain gp130/interleukin-6-receptor transducer as well as gp190/low affinity LIF receptor. For CNTF, addition of a third subunit, or alpha subunit, defines the high-affinity CNTF receptor. In the present study, we analyzed the binding interactions of LIF and CNTF in human cell lines and showed a mutual displacement for LIF and CNTF toward the trimeric high-affinity CNTF receptor. Similar results were obtained in the JEG cell line, which only expressed the gp130/gp190 high-affinity LIF receptor, by adding a soluble form of the alpha CNTF receptor to the system to reconstitute the high-affinity-type CNTF receptor. The different receptor subunits were then expressed separately in transfected cells and their binding capacities analyzed. The results showed that the heterocomplex CNTF/alpha CNTF receptor bound to gp130 with an affinity of 3-5 x 10(-10)M, whereas LIF interacted mainly with gp190. In summary, the observed competition between LIF and CNTF does not result from the binding to a common site or receptor subunit, but rather to the interaction of the three receptor components to create a conformational site common to both LIF and CNTF.

The recently cloned interleukin (IL)-11 displays many biological properties in common with those reported for IL-6. In order to analyze the nature and the functionality of the IL-11 receptor we developed a proliferative assay using the human multifactor-dependent cell line TF1. We showed that a blocking monoclonal antibody GPX7 raised against the gp130/IL-6 receptor transducing subunit was also able to inhibit the IL-11-triggered TF1 line proliferation. In addition, involvement of gp130 in IL-11 signaling was demonstrated by an induction of the transducing protein phosphorylation in response to IL-11, as observed for IL-6. In contrast, the blocking monoclonal antibody B-R6, which recognized the gp80/IL-6 binding subunit failed to interfere with the IL-11 proliferative signal in the TF1 cell line. Similarly, we did not observe any competition between IL-6 and IL-11 for a putative common binding site on the cell surface. These results suggest that the IL-11 binding component is different from the gp80/IL-6 receptor. In conclusion, IL-11, along with IL-6, leukemia inhibitory factor, oncostatin M and ciliary neurotrophic factor, belongs to the same family of cytokines, using gp130 as a transducing protein.