Treatment of HCC with claudin-1-specific antibodies suppresses carcinogenic signaling and reprograms the tumor microenvironment.

BACKGROUND & AIMS: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored. METHODS: Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC. RESULTS: Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line-derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment. CONCLUSIONS: Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC. IMPACT AND IMPLICATIONS: Hepatocellular carcinoma (HCC) is associated with high mortality and unsatisfactory treatment options. Herein, we identified the cell surface protein Claudin-1 as a treatment target for advanced HCC. Monoclonal antibodies targeting Claudin-1 inhibit tumor growth in patient-derived ex vivo and in vivo models by modulating signaling, cell stemness and the tumor immune microenvironment. Given the differentiated mechanism of action, the identification of Claudin-1 as a novel therapeutic target for HCC provides an opportunity to break the plateau of limited treatment response. The results of this preclinical study pave the way for the clinical development of Claudin-1-specific antibodies for the treatment of advanced HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.

A monoclonal antibody targeting nonjunctional claudin-1 inhibits fibrosis in patient-derived models by modulating cell plasticity.

Tissue fibrosis is a key driver of end-stage organ failure and cancer, overall accounting for up to 45% of deaths in developed countries. There is a large unmet medical need for antifibrotic therapies. Claudin-1 (CLDN1) is a member of the tight junction protein family. Although the role of CLDN1 incorporated in tight junctions is well established, the function of nonjunctional CLDN1 (njCLDN1) is largely unknown. Using highly specific monoclonal antibodies targeting a conformation-dependent epitope of exposed njCLDN1, we show in patient-derived liver three-dimensional fibrosis and human liver chimeric mouse models that CLDN1 is a mediator and target for liver fibrosis. Targeting CLDN1 reverted inflammation-induced hepatocyte profibrogenic signaling and cell fate and suppressed the myofibroblast differentiation of hepatic stellate cells. Safety studies of a fully humanized antibody in nonhuman primates did not reveal any serious adverse events even at high steady-state concentrations. Our results provide preclinical proof of concept for CLDN1-specific monoclonal antibodies for the treatment of advanced liver fibrosis and cancer prevention. Antifibrotic effects in lung and kidney fibrosis models further indicate a role of CLDN1 as a therapeutic target for tissue fibrosis across organs. In conclusion, our data pave the way for further therapeutic exploration of CLDN1-targeting therapies for fibrotic diseases in patients.

Tlr2 on Bone Marrow and Non-Bone Marrow Derived Cells Regulates Inflammation and Organ Injury in Cooperation with Tlr4 During Resuscitated Hemorrhagic Shock.

BACKGROUND: Although the role of TLR4 in driving inflammation and organ injury after hemorrhagic shock and resuscitation (H/R) is well established, the role of TLR2-another receptor for damage-associated molecular pattern (DAMP) molecules-is not. In this study, we used a combination of TLR2 and wild type (WT) mice treated with anti-TLR2 and anti-TLR4 neutralizing monoclonal antibodies (mAb) to discern the contribution of TLR2 relative to TLR4 to the systemic inflammatory response in murine H/R. MATERIAL AND METHODS: WT mice, TLR2, and WT mice receiving an anti-TLR2 or an anti-TLR4 mAB (given as a pretreatment) were sacrificed at 6 or 20 h post-H/R. Bone marrow TLR2/WT chimeric mice were created to assess the importance of immune and nonimmune cell-associated TLR2. RESULTS: TLR2 mice subjected to H/R exhibited significantly less liver damage and lower markers of systemic inflammation only at 20 h. Bone marrow chimeric mice using combinations of TLR2 mice and WT mice demonstrated that TLR2 on non-bone marrow derived cells played a dominant role in the differences at 20 h. Interestingly, WT mice treated with anti-TLR2 mAB demonstrated a reduction in organ damage and systemic inflammation at both 6 and 20 h following H/R. A combination of anti-TLR2 mAB and anti-TLR4 mAB showed that both receptors drive IP-10 and KC levels and that there is cooperation for increases in IL-6, MIG, and MCP-1 levels between TLR2 and TLR4. CONCLUSION: These data also support the conclusion that TLR2 and TLR4 act in concert as important receptors in the host immune response to H/R.

Maximizing the potency of an anti-TLR4 monoclonal antibody by exploiting proximity to Fcγ receptors.

In order to treat Toll like receptor 4 (TLR4)-mediated diseases, we generated a potent antagonistic antibody directed against human TLR4, Hu 15C1. This antibody's potency can be modulated by engaging not only TLR4 but also Fcγ receptors (FcγR), a mechanism that is driven by avidity and not cell signaling. Here, using various formats of the antibody, we further dissect the relative contributions of the Fv and Fc portions of Hu 15C1, discovering that the relationship to potency of the different antibody arms is not linear. First, as could be anticipated, we observed that Hu 15C1 co-engages up to 3 receptors on the same plasma membrane, i.e., 2 TLR4 molecules (via its variable regions) and either FcγRI or FcγRIIA (via the Fc). The Kd of these interactions are in the nM range (3 nM of the Fv for TLR4 and 47 nM of the Fc for FcγRI). However, unexpectedly, neutralization experiments revealed that, due to the low level of cell surface TLR4 expression, the avidity afforded by engagement through 2 Fv arms was significantly limited. In contrast, the antibody's neutralization capacity increases by 3 logs when able to exploit Fc-FcγR interactions. Taken together, these results demonstrate an unforeseen level of contribution by FcγRs to an antibody's effectiveness when targeting a cell surface protein of relatively low abundance. These findings highlight an exploitable mechanism by which FcγR-bearing cells may be more powerfully targeted, envisioned to be broadly applicable to other reagents aimed at neutralizing cell surface targets on cells co-expressing FcγRs.

Selective antibody intervention of Toll-like receptor 4 activation through Fc γ receptor tethering.

Inflammation is mediated mainly by leukocytes that express both Toll-like receptor 4 (TLR4) and Fc γ receptors (FcγR). Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking inflammatory cells while sparing structural cells, which are FcγR-negative, represents an elegant strategy when targeting the underlying causes of human diseases. Here, we report a novel tethering mechanism of the Fv and Fc portions of anti-TLR4 blocking antibodies that achieves increased potency on inflammatory cells. In the presence of ligand (e.g. lipopolysaccharide (LPS)), TLR4 traffics into glycolipoprotein microdomains, forming concentrated protein platforms that include FcγRs. This clustering produces a microenvironment allowing anti-TLR4 antibodies to co-engage TLR4 and FcγRs, increasing their avidity and thus substantially increasing their inhibitory potency. Tethering of antibodies to both TLR4 and FcγRs proves valuable in ameliorating inflammation in vivo. This novel mechanism of action therefore has the potential to enable selective intervention of relevant cell types in TLR4-driven diseases.

The composite cytokine p28/cytokine-like factor 1 sustains B cell proliferation and promotes plasma cell differentiation.

IL-27 is an APC-derived IL-6/IL-12 family composite cytokine with multiple functions such as regulation of Th1, Th17, and regulatory T cell differentiation, B cell proliferation, and Ig class switching. The IL-27 complex is formed by the association of the cytokine p28 with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27 cytokine and soluble receptor subunits p28 and EBI3 can be secreted independently. The p28 subunit has been shown to have IL-27-independent biological activities. We previously demonstrated that p28 can form an alternative composite cytokine with the EBI3 homolog cytokine-like factor 1 (CLF; CRLF1). p28/CLF modulates NK cell activity and CD4 T cell cytokine production in vitro. In this study we used IL-6-dependent plasmacytoma cell line B9 and CD4 T cells from IL-27Rα-deficient mice to demonstrate that p28/CLF activates IL-27-unresponsive cells, indicating that p28/CLF and IL-27 signal through different receptors. The observation that p28/CLF, unlike IL-27, sustains B9 plasmacytoma cell proliferation prompted us to investigate the effects of p28/CLF on mouse B cells. We observed that p28/CLF induces IgM, IgG2c, and IgG1 production and plasma cell differentiation. p28/CLF therefore has the potential to contribute to B and plasma cell function, differentiation, and proliferation in normal and pathological conditions such as Castelman's disease and multiple myeloma.

A polyglutamic acid motif confers IL-27 hydroxyapatite and bone-binding properties.

The p28 subunit of the composite cytokine IL-27 comprises a polyglutamic acid domain, which is unique among type I cytokines. This domain is very similar to the acidic domain known to confer hydroxyapatite (HA)-binding properties and bone tropism to bone sialoprotein. We observed IL-27 binding to HA, in accordance with previous studies reporting successful p28 HA chromatography. The IL-27 polyglutamic acid domain is located in a flexible inter-α helix loop, and HA-bound IL-27 retained biological activity. Using IL-27 alanine mutants, we observed that the p28 polyglutamic acid domain confers HA- and bone-binding properties to IL-27 in vitro and bone tropism in vivo. Because IL-27 is a potent regulator of cells residing in endosteal bone marrow niches such as osteoclasts, T regulatory, memory T, plasma, and stem cells, this specific property could be beneficial for therapeutic applications. IL-27 has potent antitumoral and antiosteoclastogenic activities. It could therefore also be useful for therapies targeting hematologic cancer or solid tumors metastasis with bone tropism. Furthermore, these observations suggest that polyglutamic motifs could be grafted onto other type I cytokine inter-α helix loops to modify their pharmacological properties.

The cytokines cardiotrophin-like cytokine/cytokine-like factor-1 (CLC/CLF) and ciliary neurotrophic factor (CNTF) differ in their receptor specificities.

Ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine (CLC) are two cytokines with neurotrophic and immunomodulatory activities. CNTF is a cytoplasmic factor believed to be released upon cellular damage, while CLC requires interaction with a soluble cytokine receptor, cytokine-like factor 1 (CLF), to be efficiently secreted. Both cytokines activate a receptor complex comprising the cytokine binding CNTF receptor α (CNTFRα) and two signaling chains namely, leukemia inhibitory factor receptor ß (LIFRß) and gp130. Human CNTF can recruit and activate an alternative receptor in which CNTFRα is substituted by IL-6Rα. As both CNTF and CLC have immune-regulatory activities in mice, we compared their ability to recruit mouse receptors comprising both gp130 and LIFRß signaling chains and either IL-6Rα or IL-11Rα which, unlike CNTFRα, are expressed by immune cells. Our results indicate that 1) mouse CNTF, like its human homologue, can activate cells expressing gp130/LIFRß with either CNTFRα or IL-6Rα and, 2) CLC/CLF is more restricted in its specificity in that it activates only the tripartite CNTFR. Several gp130 signaling cytokines influence T helper cell differentiation. We therefore investigated the effect of CNTF on CD4 T cell cytokine production. We observed that CNTF increased the number of IFN-γ producing CD4 T cells. As IFN-γ is considered a mediator of the therapeutic effect of IFN-ß in multiple sclerosis, induction of IFN-γ by CNTF may contribute to the beneficial immunomodulatory effect of CNTF in mouse multiple sclerosis models. Together, our results indicate that CNTF activates the same tripartite receptors in mouse and human cells and further validate rodent models for pre-clinical investigation of CNTF and CNTF derivatives. Furthermore, CNTF and CLC/CLF differ in their receptor specificities. The receptor α chain involved in the immunomodulatory effects of CLC/CLF remains to be identified.

IL-6 activates STAT5 in T cells.

BACKGROUND: IL-6 is a pleiotropic cytokine which emerged recently as a key regulator of CD4 T cell function. IL-6 alone or in combination with other cytokines promotes T helper 1, T helper 17 and T follicular helper cell differentiation whilst inhibiting the induction of regulatory T cell generation. IL-6 activates multiple pathways among which JAK/STAT3 is the most clearly validated in the control of CD4 T helper differentiation. Activation of STAT5 by cytokines such as IL-2 can counteract IL-6-induced T helper 17 and T follicular helper cell differentiation and promote the induction of regulatory T cell generation. STAT5 and STAT3 are known to compete for promoter binding sites in CD4 T cells and the two transcription factors are believed to have opposite functions in the control of CD4 T cell differentiation. METHODS: We analyzed IL-6-induced STAT1, 3 and 5 activation by flow cytometry (phosflow) in mouse mononuclear cells and its effect on the level of the mRNA coding for cytokine-inducible SH2-containing protein (CIS). RESULTS: The results show that IL-6 also induces STAT5 activation in both CD4 and CD8 T as well as NK cells. Analysis of STAT5 phosphorylation in CD4 T cells indicates that it is transient and requires higher cytokine concentrations than that of STAT3. CD4 T cell stimulation with IL-6 induces the synthesis of CIS, which is encoded by a gene known to be regulated by STAT5. CONCLUSIONS: Thus, IL-6 at concentrations corresponding to levels observed in the serum during inflammation may activate, in CD4 T cells, a STAT5-negative feedback loop which alters the balance between STAT3-dependent pro-inflammatory helper T cells and STAT5-induced T regulatory cells. STAT5 activation may modulate the differentiation of T helper cells through attenuation of TGF-ß stability and production. Since STAT5 is directly activated by Janus kinases, therapeutic approaches designed to inhibit STAT3 activation or to recruit STAT3 phosphatases may be useful in altering the balance of activated STAT3 and STAT5 in favor a profile that would be beneficial in pathologies involving IL-6.

Constitutive activation of epithelial TLR4 augments inflammatory responses to mucosal injury and drives colitis-associated tumorigenesis.

BACKGROUND: Chronic intestinal inflammation culminates in cancer and a link to Toll-like receptor-4 (TLR4) has been suggested by our observation that TLR4 deficiency prevents colitis-associated neoplasia. In the current study we address the effect of the aberrant activation of epithelial TLR4 on induction of colitis and colitis-associated tumor development. We take a translational approach to address the consequences of increased TLR signaling in the intestinal mucosa. METHODS: Mice transgenic for a constitutively active TLR4 under the intestine-specific villin promoter (villin-TLR4 mice) were treated with dextran sodium sulfate (DSS) for acute colitis and azoxymethane (AOM)-DSS TLR4 expression was analyzed by immunohistochemistry in colonic tissue from patients with ulcerative colitis (UC) and UC-associated cancer. The effect of an antagonist TLR4 antibody (Ab) was tested in prevention of colitis-associated neoplasia in the AOM-DSS model. RESULTS: Villin-TLR4 mice were highly susceptible to both acute colitis and colitis-associated neoplasia. Villin-TLR4 mice had increased epithelial expression of COX-2 and mucosal PGE2 production at baseline. Increased severity of colitis in villin-TLR4 mice was characterized by enhanced expression of inflammatory mediators and increased neutrophilic infiltration. In human UC samples, TLR4 expression was upregulated in almost all colitis-associated cancer and progressively increased with grade of dysplasia. As a proof of principle, a TLR4/MD-2 antagonist antibody inhibited colitis-associated neoplasia in the mouse model. CONCLUSIONS: Our results show that regulation of TLRs can affect the outcome of both acute colitis and its consequences, cancer. Targeting TLR4 and other TLRs may ultimately play a role in prevention or treatment of colitis-associated cancer.

Toll-like receptor 4 signaling by follicular dendritic cells is pivotal for germinal center onset and affinity maturation.

Germinal centers (GCs) are specialized microenvironments where antigen-activated B cells undergo proliferation, immunoglobulin (Ig) class switch recombination, somatic hypermutation (SHM), and affinity maturation. Within GCs, follicular dendritic cells (FDCs) are key players in driving these events via direct interaction with GC B cells. Here, we provide in vivo evidence that FDCs express and upregulate Toll-like-receptor (TLR) 4 in situ during germinal center reactions, confirm that their maturation is driven by TLR4, and associate the role of FDC-expressed TLR4 with quantitative and qualitative affects of GC biology. In iterative cycles of predictions by in silico modeling subsequently verified by in vivo experiments, we demonstrated that TLR4 signaling modulates FDC activation, strongly impacting SHM and generation of Ig class-switched high-affinity plasma and memory B cells. Thus, our data place TLR4 in the heart of adaptive humoral immunity, providing further insight into mechanisms driving GCs arising in both health and disease.

A novel Toll-like receptor 4 antagonist antibody ameliorates inflammation but impairs mucosal healing in murine colitis.

Dysregulated innate immune responses to commensal bacteria contribute to the development of inflammatory bowel disease (IBD). TLR4 is overexpressed in the intestinal mucosa of IBD patients and may contribute to uncontrolled inflammation. However, TLR4 is also an important mediator of intestinal repair. The aim of this study is to examine the effect of a TLR4 antagonist on inflammation and intestinal repair in two murine models of IBD. Colitis was induced in C57BL/6J mice with dextran sodium sulfate (DSS) or by transferring CD45Rb(hi) T cells into RAG1-/- mice. An antibody (Ab) against the TLR4/MD-2 complex or isotype control Ab was administered intraperitoneally during DSS treatment, recovery from DSS colitis, or induction of colitis in RAG1-/- mice. Colitis severity was assessed by disease activity index (DAI) and histology. The effect of the Ab on the inflammatory infiltrate was determined by cell isolation and immunohistochemistry. Mucosal expression of inflammatory mediators was analyzed by real-time PCR and ELISA. Blocking TLR4 at the beginning of DSS administration delayed the development of colitis with significantly lower DAI scores. Anti-TLR4 Ab treatment decreased macrophage and dendritic cell infiltrate and reduced mucosal expression of CCL2, CCL20, TNF-alpha, and IL-6. Anti-TLR4 Ab treatment during recovery from DSS colitis resulted in defective mucosal healing with lower expression of COX-2, PGE(2), and amphiregulin. In contrast, TLR4 blockade had minimal efficacy in ameliorating inflammation in the adoptive transfer model of chronic colitis. Our findings suggest that anti-TLR4 therapy may decrease inflammation in IBD but may also interfere with colonic mucosal healing.

Increased release of sMD-2 during human endotoxemia and sepsis: a role for endothelial cells.

MD-2 is the crucial cofactor of TLR4 in the detection of LPS. Here, we show that soluble MD-2 (sMD-2) circulates in plasma of healthy individuals as a polymeric protein. The total amount of sMD-2 in septic plasma was strongly elevated and contained both sMD-2 polymers and monomers, the latter representing the putative biologically active form of MD-2. Moreover, during experimental human endotoxemia, the monomeric and total sMD-2 content in plasma increased with the kinetics of an acute phase protein. The increase in sMD-2 monomers was paralleled by enhanced TLR4 costimulatory activity. The presence of functional sMD-2 during endotoxemia and sepsis was confirmed by immunodepletion. Immunohistochemistry revealed that MD-2 expression in septic patients was strongly enhanced on endothelium and multiple inflammatory cells in lung and liver. In vitro studies showed that sMD-2 release appears to be restricted to endothelial cells and dendritic cells. Release of sMD-2 by endothelial cells was strongly enhanced by LPS and TNF-alpha stimulation. Taken together, this study demonstrates the increase of both circulating polymeric and functional monomeric sMD-2 during endotoxemia and sepsis, and evidence is provided that the endothelium is involved in this process.

Soluble MD-2 is an acute-phase protein and an opsonin for Gram-negative bacteria.

Myeloid differentiation factor-2 (MD-2) is a lipopolysaccharide (LPS)-binding protein usually coexpressed with and binding to Toll-like receptor 4 (TLR4), conferring LPS responsiveness of immune cells. MD-2 is also found as a soluble protein. Soluble MD-2 (sMD-2) levels are markedly elevated in plasma from patients with severe infections, and in other fluids from inflamed tissues. We show that sMD-2 is a type II acute-phase protein. Soluble MD-2 mRNA and protein levels are up-regulated in mouse liver after the induction of an acute-phase response. It is secreted by human hepatocytic cells and up-regulated by interleukin-6. Soluble MD-2 binds to Gram-negative but not Gram-positive bacteria, and sMD-2 secreted by hepatocytic cells is an essential cofactor for the activation of TLR4-expressing cells by Gram-negative bacteria. Soluble MD-2 opsonization of Gram-negative bacteria accelerates and enhances phagocytosis, principally by polymorphonuclear neutrophils. In summary, our results demonstrate that sMD-2 is a newly recognized type II acute-phase reactant, an opsonin for Gram-negative bacteria, and a cofactor essential for the activation of TLR4-expressing cells. This suggests that sMD-2 plays a key role in the host innate immune response to Gram-negative infections.

Interleukin-6 biology is coordinated by membrane-bound and soluble receptors: role in inflammation and cancer.

Cytokine receptors, which exist in membrane-bound and soluble forms, bind their ligands with comparable affinity. Although most soluble receptors are antagonists and compete with their membrane-associated counterparts for the ligands, certain soluble receptors are agonists. In these cases, complexes of ligand and soluble receptor bind on target cells to second receptor subunits and initiate intracellular signaling. The soluble receptors of the interleukin (IL)-6 family of cytokines (sIL-6R, sIL-11R, soluble ciliary neurotrophic factor receptor) are agonists capable of transmitting signals through interaction with the universal signal-transducing receptor for all IL-6 family cytokines, gp130. In vivo, the IL-6/sIL-6R complex stimulates several types of cells, which are unresponsive to IL-6 alone, as they do not express the membrane IL-6R. We have named this process trans-signaling. The generation of soluble cytokine receptors occurs via two distinct mechanisms-limited proteolysis and translation-from differentially spliced mRNA. We have demonstrated that a soluble form of the IL-6 family signaling receptor subunit gp130, which is generated by differential splicing, is the natural inhibitor of IL-6 trans-signaling responses. We have shown that in many chronic inflammatory diseases, including chronic inflammatory bowel disease, peritonitis, rheumatoid arthritis, asthma, as well as colon cancer, IL-6 trans-signaling is critically involved in the maintenance of a disease state, by promoting transition from acute to chronic inflammation. Moreover, in all these models, the course of the disease can be disrupted by specifically interfering with IL-6 trans-signaling using the soluble gp130 protein. The pathophysiological mechanisms by which the IL-6/sIL-6R complex regulates the inflammatory state are discussed.

Detection of epsilon class switching and IgE synthesis in human B cells.

We observed that mast cells, as other cells expressing the CD40 ligand CD154, can trigger IgE synthesis in B cells in the presence of interleukin (IL)-4. Numerous complementary techniques can be used to follow the succession of molecular events leading to IgE synthesis. This chapter will illustrate how human B cells (naïve or memory) can be purified, stored, and cultivated in medium that is permissive for IgE synthesis and stimulated with IL-4 or IL-13 and CD40 activation, the latter being induced by soluble CD154, anti-CD40 antibodies, or CD154-expressing cells. All these molecules are expressed by mast cells. The quantification of the epsilon-sterile transcript synthesis by polymerase chain reaction or Northern blot, the epsilon excision circles produced during immunoglobulin heavy chain locus rearrangement by polymerase chain reaction, and the IgE production by enzyme-linked immunosorbent assay will be described.

clc is co-expressed with clf or cntfr in developing mouse muscles.

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.

Expression of biologically active mouse ciliary neutrophic factor (CNTF) and soluble CNTFRalpha in Escherichia coli and characterization of their functional specificities.

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.

Two different contact sites are recruited by cardiotrophin-like cytokine (CLC) to generate the CLC/CLF and CLC/sCNTFRalpha composite cytokines.

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.

Functionally active fusion protein of the novel composite cytokine CLC/soluble CNTF receptor.

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.