The alpha 5 gene product assembles with multiple acetylcholine receptor subunits to form distinctive receptor subtypes in brain.

The acetylcholine receptor (AChR) alpha 5 gene has been classified as a member of the AChR gene family based on sequence homology. Expression studies, however, have yet to identify a function for the alpha 5 gene product or even to demonstrate an interaction with known AChR subunits. We report here that the alpha 5 gene product is identical to the 49 kd protein previously found on immunoblots of AChRs purified from brain and ciliary ganglia. In brain the alpha 5 gene product is present both in alpha 3- and in alpha 4-based receptor subtypes, while in the ganglion it is found in an alpha 3-based receptor subtype concentrated in postsynaptic membrane. Immunoprecipitation experiments with subunit-specific monoclonal antibodies indicate that some native AChRs are likely to have at least three kinds of subunits, with two being of the alpha type. These findings support new views about the construction of AChRs in neurons.

Neurons assemble acetylcholine receptors with as many as three kinds of subunits while maintaining subunit segregation among receptor subtypes.

A family of genes encoding neuronal acetylcholine receptor (AChR) subunits has been identified and cloned from vertebrates. Expression studies have implied that as few as one or two kinds of subunits may be sufficient to construct neuronal AChRs and that multiple pair-wise combinations of the gene products are capable of generating functional receptors. We show here that a class of AChRs with a predominantly synaptic location on neurons contains receptors having at least three types of subunits and that the subunits are encoded by the alpha 3, beta 4, and alpha 5 AChR genes. In addition, we show that a class of extrasynaptic AChRs on the same neurons contains the alpha 7 subunits but lacks the alpha 3, beta 4, and alpha 5 subunits. The results demonstrate that native AChRs on neurons are more complex in composition than previously appreciated and suggest that constraints on subunit interactions limit the kinds of receptor species produced.

Canine mesenchymal stem cells are neurotrophic and angiogenic: An in vitro assessment of their paracrine activity.

Mesenchymal stem cells (MSCs) have been used in cell replacement therapies for connective tissue damage, but also can stimulate wound healing through paracrine activity. In order to further understand the potential use of MSCs to treat dogs with neurological disorders, this study examined the paracrine action of adipose-derived canine MSCs on neuronal and endothelial cell models. The culture-expanded MSCs exhibited a MSC phenotype according to plastic adherence, cell morphology, CD profiling and differentiation potential along mesenchymal lineages. Treating the SH-SY5Y neuronal cell line with serum-free MSC culture-conditioned medium (MSC CM) significantly increased SH-SY5Y cell proliferation (P <0.01), neurite outgrowth (P = 0.0055) and immunopositivity for the neuronal marker ßIII-tubulin (P = 0.0002). Treatment of the EA.hy926 endothelial cell line with MSC CM significantly increased the rate of wound closure in endothelial cell scratch wound assays (P = 0.0409), which was associated with significantly increased endothelial cell proliferation (P <0.05) and migration (P = 0.0001). Furthermore, canine MSC CM induced endothelial tubule formation in EA.hy926 cells in a soluble basement membrane matrix. Hence, this study has demonstrated that adipose-derived canine MSC CM stimulated neuronal and endothelial cells probably through the paracrine activity of MSC-secreted factors. This supports the use of canine MSC transplants or their secreted products in the clinical treatment of dogs with neurological disorders and provides some insight into possible mechanisms of action.

Production and characterization of monoclonal antibodies against the leukemia inhibitory factor low affinity receptor, gp190.

Leukemia inhibitory factor (LIF), oncostatin-M (OSM), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT1) act through transmembrane receptors which share the gp190 glycoprotein chain. The understanding of its involvement in the biology of these cytokines is of importance since these systems have recently been shown to participate in major inflammatory and neoplastic processes such as myelomatosis (Rose-John, S., Heinrich, P.C., 1994. Soluble receptors for cytokines and growth factors: generation and biological function. Biochem. J. 300, 281). In addition, this family of receptors also shares the gp130 transducing chain, with the IL6 and IL11 receptors. Because IL6 and gp130 were the first members to be discovered, most of the available reagents are directed at them. In this respect, monoclonal antibodies have played a major role in elucidating these receptor/ligand interactions and exploring the pathophysiological aspects of their biology. So far, no such reagents have been described for the gp190. We now report the production and characterization of 16 monoclonal antibodies directed against human gp190. They were obtained using recombinant chimeric or truncated proteins produced in a eukaryotic CHO cell line. One was able to block the biological activity of LIF. Because gp190 comprises two hematopoietin binding domains, crude epitope mapping was possible using the same reagents. While more of these antibodies are required, the present set validate the technological approach used for their preparation and should improve our understanding of this class of cytokines.

Pro-inflammatory cytokines IL6, TNF-alpha, IL1beta, procalcitonin, lipopolysaccharide binding protein and C-reactive protein in infective endocarditis.

OBJECTIVE: To evaluate the serum levels and diagnostic value of cytokines and acute phase proteins in patients with infective endocarditis (IE). PATIENTS AND METHODS: Serum samples from 63 patients diagnosed with IE and 71 control patients were analysed for the following markers: interleukin-6 (IL6), tumour necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL1beta), procalcitonin (PCT), lipopolysaccharide binding protein (LBP) and C-reactive protein (CRP). RESULTS: Serum levels of IL6, IL1beta and CRP were significantly elevated in patients with IE as compared to controls. PCT, TNF-alpha and LBP were not elevated. CONCLUSION: Serum CRP and IL6 are elevated in IE. IL 6 may aid in establishing the diagnosis. There was no correlation between IL 6 levels and CRP, causative microorganism, echocardiographic features or outcome.

An antagonist for the leukemia inhibitory factor receptor inhibits leukemia inhibitory factor, cardiotrophin-1, ciliary neurotrophic factor, and oncostatin M.

The leukemia inhibitory factor receptor (LIF-R) is activated not only by LIF, but also by cardiotrophin-1, ciliary neurotrophic factor with its receptor, and oncostatin M (OSM). Each of these cytokines induces the hetero-oligomerization of LIF-R with gp130, a signal-transducing subunit shared with interleukin-6 and interleukin-11. The introduction of mutations into human LIF that reduced the affinity for gp130 while retaining affinity for LIF-R has generated antagonists for LIF. In the current study, a LIF antagonist that was free of detectable agonistic activity was tested for antagonism against the family of LIF-R ligands. On cells that express LIF-R and gp130, all LIF-R ligands were antagonized. On cells that also express OSM receptor, OSM was not antagonized, demonstrating that the antagonist is specific for LIF-R. Ligand-triggered tyrosine phosphorylation of both LIF-R and gp130 was blocked by the antagonist. The antagonist is therefore likely to work by preventing receptor oligomerization.

Characterization of the receptor binding sites of human leukemia inhibitory factor and creation of antagonists.

Residues in human leukemia inhibitory factor (hLIF) crucial for binding to both the human LIF receptor (R) and gp130 were identified by analysis of alanine scanning mutants of hLIF in assays for both receptor binding and bioactivity. The region of hLIF most important for binding to the hLIF-R is composed of residues from the amino terminus of the D-helix, carboxyl terminus of the B-helix, and C-D loop. This site forms a distinct surface at the end of the four-helix bundle in the tertiary structure of the closely related murine LIF. The two residues of hLIF that contribute the majority of free energy for hLIF-R binding, Phe-156 and Lys-159 are surrounded by other residues which have only a moderate impact. This arrangement of a few key residues surrounded by less important ones is analogous to the functional binding epitope of human growth hormone for its receptor. A second region of hLIF that includes residues from the carboxyl terminus of the D-helix and A-B loop also had a weak influence on hLIF-R binding. Residues in hLIF from both the A- and C-helices are involved in binding the gp130 co-receptor. Abolition of the gp130 binding site in hLIF created antagonists of LIF action.

Leukaemia inhibitory factor is retrogradely transported by a distinct population of adult rat sensory neurons: co-localization with trkA and other neurochemical markers.

Sciatic sensory afferents that retrogradely transport and accumulate leukaemia inhibitory factor (LIF) within their soma were characterized in the adult rat in vivo. Twenty-four percent of neurons within the L4 and L5 dorsal root ganglia accumulated biotinylated LIF following intraneural injection of the cytokine into the sciatic nerve. Labelled cell bodies were predominantly of small diameter (20.1 +/- 0.5 microm). Retrograde transport was eliminated by excess unlabelled LIF but not by the related cytokines, ciliary-derived neurotrophic factor (CNTF) and interleukin-6 (IL-6). Double labelling revealed that the majority (81%) of LIF-accumulating neurons were immunopositive for CGRP and 34% were immunopositive for the cell surface glycoconjugate IB4. Sixty-two percent of LIF-accumulating neurons were immunopositive for trkA. Our results demonstrate a group of small-diameter sensory neurons that retrogradely transport LIF, comprising cells that constitutively express neuropeptides and those likely to be peptide-deficient. LIF-accumulating neurons expressing trkA are also potentially responsive to nerve growth factor. It is likely that the LIF-accumulating neurons described in this study are nociceptive in function.

Müller-cell-derived leukaemia inhibitory factor arrests rod photoreceptor differentiation at a postmitotic pre-rod stage of development.

In the present study, we examine rod photoreceptor development in dissociated-cell cultures of neonatal mouse retina. We show that, although very few rhodopsin+ rods develop in the presence of 10% foetal calf serum (FCS), large numbers develop in the absence of serum, but only if the cell density in the cultures is high. The rods all develop from nondividing rhodopsin- cells, and new rods continue to develop from rhodopsin- cells for at least 6-8 days, indicating that there can be a long delay between when a precursor cell withdraws from the cell cycle and when it becomes a rhodopsin+ rod. We show that FCS arrests rod development in these cultures at a postmitotic, rhodopsin-, pre-rod stage. We present evidence that FCS acts indirectly by stimulating the proliferation of Müller cells, which arrest rod differentiation by releasing leukaemia inhibitory factor (LIF). These findings identify an inhibitory cell-cell interaction, which may help to explain the long delay that can occur both in vitro and in vivo between cell-cycle withdrawal and rhodopsin expression during rod development.

The crystal structure and biological function of leukemia inhibitory factor: implications for receptor binding.

The structure of murine leukemia inhibitory factor (LIF) has been determined by X-ray crystallography at 2.0 A resolution. The main chain fold conforms to the four alpha-helix bundle topology previously observed for several members of the hematopoietic cytokine family. Of these, LIF shows closest structural homology to granulocyte colony-stimulating factor and growth hormone (GH). Sequence alignments for the functionally related molecules oncostatin M and ciliary neurotrophic factor, when mapped to the LIF structure, indicate regions of conserved surface character. Analysis of the biological function and receptor specificity of a series of human-mouse LIF chimeras implicate two regions of receptor interaction that are located in the fourth helix and the preceding loop. A model for receptor binding based on the structure of the GH ligand-receptor complex requires additional, novel features to account for these data.

Mediation of interleukin-11-dependent biological responses by a soluble form of the interleukin-11 receptor.

Interleukin-11 (IL-11) is a polyfunctional cytokine whose biological actions require a specific IL-11 receptor (IL-11R) and the transmembrane transducer gp130. Here we report the production of a soluble form of the murine IL-11R and demonstrate that it interacts with IL-11 ligand with high affinity. The affinity of IL-11 alone for gp130 is below the level of detection, but a complex of IL-11 and soluble IL-11R interacts with gp130 with high affinity. The addition of soluble IL-11R potentiates the effects of exogenous IL-11 in cells that are normally responsive to IL-11. A biological response to IL-11 can be reconstituted in BAF cells transfected with gp130 by addition of IL-11 and soluble IL-11R. These findings show that the cytoplasmic domain of the IL-11R is not required for the biological effects of IL-11 and that a complex of IL-11 and IL-11R mediates signalling by association with gp130.

Cardiotrophin-1 activates a distinct form of cardiac muscle cell hypertrophy. Assembly of sarcomeric units in series VIA gp130/leukemia inhibitory factor receptor-dependent pathways.

Cardiotrophin-1 (CT-1) was recently isolated by expression cloning based on its ability to induce an increase in cell size in neonatal rat ventricular cardiomyocytes. Sequence similarity data suggested that CT-1 is a novel member of a family of structurally related cytokines sharing the receptor component gp130. The present study documents that gp130 is required for CT-1 signaling in cardiomyocytes, by demonstrating that a monoclonal anti-gp130 antibody completely inhibits c-fos induction by CT-1. Similarly, a leukemia inhibitory factor receptor subunit beta (LIFRbeta) antagonist effectively blocks the CT-1 induction of c-fos, indicating a requirement for LIFRbeta in the hypertrophic response, as well. Upon stimulation with CT-1, both gpl30 and the LIFRbeta are tyrosine-phosphorylated, providing further evidence that CT-1 signals through the gp130/LIFRbeta heterodimer in cardiomyocytes. CT-1 induces a hypertrophic response in cardiomyocytes that is distinct from the phenotype seen after alpha-adrenergic stimulation, both with regard to cell morphology and gene expression pattern. Stimulation with CT-1 results in an increase in cardiac cell size that is characterized by an increase in cell length but no significant change in cell width. Confocal laser microscopy of CT-1 stimulated cells reveals the assembly of sarcomeric units in series rather than in parallel, as seen after alpha-adrenergic stimulation. CT-1 induces a distinct pattern of immediate early genes, and up-regulates the atrial natriuretic factor (ANF) gene, but does not affect skeletal alpha-actin or myosin light chain-2v expression. As evidenced by nuclear run-on transcription assays, both CT-1 and alpha-adrenergic stimulation lead to an increase in ANF gene transcription. Transient transfection analyses document that, in contrast to alpha-adrenergic stimulation, the CT-1 responsive cis-regulatory elements are located outside of the proximal 3 kilobase pairs of the ANF 5'-flanking region. These studies indicate that CT-1 can activate a distinct form of myocardial cell hypertrophy, characterized by the promotion of sarcomere assembly in series, via gpl30/LIFRbeta-dependent signaling pathways.

Leukemia inhibitory factor and its receptor promote adipocyte differentiation via the mitogen-activated protein kinase cascade.

Extracellular factors and intracellular signaling pathways involved in early events of adipocyte differentiation are poorly defined. It is shown herein that expression of leukemia inhibitory factor (LIF) and LIF receptor is developmentally regulated during adipocyte differentiation. Preadipocytes secrete bioactive LIF, and an antagonist of LIF receptor inhibits adipogenesis. Genetically modified embryonic stem (ES) cells combined with culture conditions to commit stem cells into the adipocyte lineage were used to examine the requirement of LIF receptor during in vitro development of adipose cells. The capacity of embryoid bodies derived from lifr(-/-) ES cells to undergo adipocyte differentiation is dramatically reduced. LIF addition stimulates adipocyte differentiation of Ob1771 and 3T3-F442A preadipocytes and that of peroxisome proliferator-activated receptor gamma2 ligand-treated mouse embryonic fibroblasts. Expression of the early adipogenic transcription factors C/EBPbeta and C/EBPdelta is rapidly stimulated following exposure of preadipose cells to LIF. The selective inhibitors of mitogen-activated protein kinase kinase, i.e. PD98059 and U0126, inhibit LIF-induced C/EBP gene expression and prevent adipocyte differentiation induced by LIF. These results are in favor of a model that implicates stimulation of LIF receptor in the commitment of preadipocytes to undergo terminal differentiation by controlling the early expression of C/EBPbeta and C/EBPdelta genes via the mitogen-activated protein kinase cascade.

Synergistic effects of glycoprotein 130 binding cytokines in combination with interleukin-1 on cartilage collagen breakdown.

OBJECTIVE: To determine whether other glycoprotein 130 (gp130) binding cytokines can mimic the effects of oncostatin M (OSM) in acting synergistically with interleukin-1alpha (IL-1alpha) to induce cartilage collagen breakdown and collagenase expression, and to determine which receptors mediate these effects. METHODS: The release of collagen and proteoglycan was assessed in bovine and human cartilage explant cultures. Messenger RNA (mRNA) and protein production from immortalized human chondrocytes (T/C28a4) was analyzed by Northern blotting and specific enzyme-linked immunosorbent assays. Collagenase activity was measured by bioassay. Cell surface receptors were detected by flow cytometry. RESULTS: OSM in combination with IL-1alpha caused a rapid synergistic induction of matrix metalloproteinase 1 mRNA, which was sustained over a 72-hour period. Flow cytometric analyses detected both the OSM-specific receptor and the gp130 receptor at the chondrocyte cell surface, but failed to detect the leukemia inhibitory factor receptor (LIFR). Cartilage degradation assays revealed that, of the gp130 binding cytokines, only OSM and IL-6, in the presence of its soluble receptor (sIL-6R), were able to act synergistically with IL-1alpha to promote collagen release. CONCLUSION: This study demonstrates that IL-6 can mimic OSM in synergizing with IL-1alpha to induce chondrocyte-mediated cartilage collagen breakdown and collagenase production. In order to have this effect, IL-6 requires the presence of its soluble receptor. The apparent absence of LIFR explains why other gp130 binding cytokines do not act in synergy with IL-1alpha. Since OSM, IL-6, and sIL-6R levels have all been shown to be elevated in the rheumatoid joint, our findings suggest that these cytokines may be key mediators of cartilage collagen catabolism in the inflammatory arthritides.