Human interleukin 4 receptor confers biological responsiveness and defines a novel receptor superfamily.

IL-4, a pleiotropic cytokine produced by T lymphocytes, plays an important role in immune responsiveness by regulating proliferation and differentiation of a variety of lymphoid and myeloid cells via binding to high affinity receptors. In this report we describe the isolation and functional expression of a human IL-4-R cDNA. When transfected into COS-7 cells, the cDNA encodes a 140-kD cell-surface protein. After transfection into a murine T cell line, the cDNA encodes a protein that binds human IL-4 with high affinity and can confer responsiveness to human IL-4. The predicted extracellular domain of the IL-4-R exhibits significant amino acid sequence homology with the beta subunit of the IL-2-R (p75), and the receptors for IL-6, erythropoietin, and prolactin. These receptors comprise a novel superfamily with extracellular domains characterized by four conserved cysteine residues and a double tryptophan-serine (WSXWS) motif located proximal to the transmembrane region.

The IL-6 signal transducer, gp130: an oncostatin M receptor and affinity converter for the LIF receptor.

Leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) are multifunctional cytokines with many similar activities. LIF is structurally and functionally related to another cytokine, Oncostatin M (OSM), that binds to the high-affinity LIF receptor but not to the low-affinity LIF receptor. A complementary DNA was isolated that encodes the high-affinity converting subunit of the LIF receptor. The converter conferred high-affinity binding of both LIF and OSM when expressed with the low-affinity LIF receptor and is identical to the signal transducing subunit of the IL-6 receptor, gp130. The gp130 subunit alone confers low-affinity binding of OSM when expressed in COS-7 cells. This receptor system resembles the high-affinity receptors for granulocyte-macrophage colony-stimulating factor, IL-3, and IL-5, which share a common subunit.

Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39.

Excessive platelet accumulation and recruitment, leading to vessel occlusion at sites of vascular injury, present major therapeutic challenges in cardiovascular medicine. Endothelial cell CD39, an ecto-enzyme with ADPase and ATPase activities, rapidly metabolizes ATP and ADP released from activated platelets, thereby abolishing recruitment. Therefore, a soluble form of CD39, retaining nucleotidase activities, would constitute a novel antithrombotic agent. We designed a recombinant, soluble form of human CD39, and isolated it from conditioned media from transiently transfected COS-1 cells and from stably transfected Chinese hamster ovary (CHO) cells. Conditioned medium from CHO cells grown under serum-free conditions was subjected to anti-CD39 immunoaffinity column chromatography, yielding a single approximately 66-kD protein with ATPase and ADPase activities. Purified soluble CD39 blocked ADP-induced platelet aggregation in vitro, and inhibited collagen-induced platelet reactivity. Kinetic analyses indicated that, while soluble CD39 had a Km for ADP of 5.9 microM and for ATP of 2.1 microM, the specificity constant kcat/Km was the same for both substrates. Intravenously administered soluble CD39 remained active in mice for an extended period of time, with an elimination phase half-life of almost 2 d. The data indicate that soluble CD39 is a potential therapeutic agent for inhibition of platelet-mediated thrombotic diatheses.

Organization of the murine and human interleukin-7 receptor genes: two mRNAs generated by differential splicing and presence of a type I-interferon-inducible promoter.

To better understand the regulation of interleukin-7 receptor (IL-7R) expression, we have pursued a detailed analysis of the structure of the murine and human IL-7R genes. The genes consist of eight exons, the sizes of which are conserved in mouse and human cells, spread out over 24 kbp (murine) and 19 kbp (human). A differential splicing event results in an mRNA encoding a secreted form of the human IL-7R gene. Primer extension and S1 nuclease analysis show a single transcriptional start site for the murine IL-7R gene. The 5'-flanking region of the murine IL-7R gene contains TATA- and CAAT-like sequences. The promoter region also contains a functional interferon regulatory element, to which the interferon-induced nuclear factors IRF-1 and IRF-2 are capable of binding and which is able to confer interferon-inducible expression on a heterologous gene. There are also potential binding sites for the transcription factors AP-1 and AP-2 as well as multiple glucocorticoid response elements. A fusion gene containing 2.5 kb of murine IL-7R 5' regulatory sequence linked to the bacterial chloramphenicol acetyltransferase gene directed expression of chloramphenicol acetyltransferase activity in murine pre-B-cell line 70Z/3 but not in the mouse fibroblast cell line NIH 3T3. Comparison of the murine and human IL-7R exon/intron boundaries with those of other hematopoietin receptor superfamily members whose exon/intron boundaries are also known reveals a conserved evolutionary structure.

Leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp130.

Leukemia inhibitory factor (LIF) is a cytokine with a broad range of activities that in many cases parallel those of interleukin-6 (IL-6) although LIF and IL-6 appear to be structurally unrelated. A cDNA clone encoding the human LIF receptor was isolated by expression screening of a human placental cDNA library. The LIF receptor is related to the gp130 'signal-transducing' component of the IL-6 receptor and to the G-CSF receptor, with the transmembrane and cytoplasmic regions of the LIF receptor and gp130 being most closely related. This relationship suggests a common signal transduction pathway for the two receptors and may help to explain similar biological effects of the two ligands. Murine cDNAs encoding soluble LIF receptors were isolated by cross-hybridization and share 70% amino acid sequence identity to the human sequence.

The murine interleukin-4 receptor: molecular cloning and characterization of secreted and membrane bound forms.

Receptors for interleukin-4 (IL-4) are expressed at low levels on a wide variety of primary cells and cultured cell lines. Fluorescence-activated sorting of CTLL-2 cells resulted in the isolation of a subclone, CTLL 19.4, which expressed 10(6) IL-4 receptors per cell. These cells were used for the purification of IL-4 receptor protein and to prepare a hybrid-subtracted cDNA probe for isolation of cDNA clones. Three classes of IL-4 receptor cDNA were identified. The first encoded a 140 kd membrane bound IL-4 receptor containing extracellular, transmembrane, and cytoplasmic domains. The second class lacked the cytoplasmic region, and the third encoded a secreted form of the receptor. All cDNA clones expressed in COS-7 cells had IL-4 binding properties comparable to the native IL-4 receptor. The soluble form of the IL-4 receptor blocked the ability of IL-4 to induce CTLL cell proliferation and may represent a regulatory molecule specific for IL-4-dependent immune responses.

Molecular cloning of a ligand for the inducible T cell gene 4-1BB: a member of an emerging family of cytokines with homology to tumor necrosis factor.

4-1BB is an inducible T cell antigen that shows sequence homology to members of an emerging family of cytokine receptors, including those for tumor necrosis factor and nerve growth factor. To aid in the analysis of the function of 4-1BB we have utilized a soluble form of the molecule as a probe to identify and clone the gene which encodes its ligand. The ligand for 4-1BB is a type II membrane glycoprotein that has homology to tumor necrosis factor, lymphotoxin, and the ligands for CD40 and CD27, all of which are themselves ligands to receptors in this superfamily. The gene for 4-1BB is on mouse chromosome 4 and maps close to the p80 form of the tumor necrosis factor receptor as well as the gene for CD30. The gene for 4-1BB ligand maps to mouse chromosome 17, but considerably distal to the tumor necrosis factor and lymphotoxin genes. Interaction of 4-1BB with its ligand induces the proliferation of activated thymocytes and splenic T cells, a response which is mimicked on similar cell populations stimulated with an antibody to 4-1BB.

Molecular and biological characterization of a murine ligand for CD40.

The CD40 surface molecule is a 277-amino-acid glycoprotein expressed on B lymphocytes, epithelial cells and some carcinoma cell lines. Monoclonal antibodies against CD40 mediate a variety of effects on B lymphocytes, including induction of intercellular adhesion, short- and long-term proliferation, differentiation and enhanced tyrosine phosphorylation of proteins. In addition, germinal centre centrocytes are prevented from undergoing apoptosis by activation through CD40 and receptor for antigen. These data indicate that CD40 could be a receptor for an unknown ligand with important functions in B-cell development and activation. This hypothesis is strengthened by the homology of the extracellular region of the CD40 molecule with a family of cell-surface glycoproteins that includes the receptors for nerve growth factor and tumour necrosis factor. Here we report the cloning of a ligand for CD40 that is expressed on the cell surface of activated T cells and mediates B-cell proliferation in the absence of co-stimulus, as well as IgE production in the presence of interleukin-4.

Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms.

We have previously reported the identification of a novel mast cell growth factor (MGF) that was shown to be a ligand for c-kit and is encoded by a gene that maps near the steel locus on mouse chromosome 10. We now report the cloning of cDNAs encoding the MGF protein. The MGF protein encoded by this cDNA can be expressed in a biologically active form as either a membrane bound protein or as a soluble factor. The soluble protein promotes the proliferation of MGF-responsive cell lines and, in the presence of erythropoietin, stimulates the formation of macroscopic [corrected] erythroid and multilineage hematopoietic colonies.