Expression cloning of a human Fc receptor for IgA.

IgA, the predominant isotype in secretions, mediates the neutralization and removal of environmental antigens from mucosal sites. Although cell surface receptors for the Fc region of IgA (Fc alpha R) have been implicated in a variety of immune effector mechanisms, the molecular features of Fc alpha R remain only marginally characterized. In this report, we describe the isolation of a clone from a myeloid cell line cDNA library that directs the expression of a cell surface molecule with IgA binding specificity. The cDNA encodes a peptide of Mr 30,000 including a putative transmembrane region with features atypical of conventional membrane-anchored proteins. Databank searches indicate that the human myeloid cell Fc alpha R sequence is unique, is a member of the immunoglobulin gene superfamily, and is related to Fc receptors for IgG (Fc gamma RI, II, and III) and IgE (Fc epsilon RI).

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.

Expression cloning of a human granulocyte colony-stimulating factor receptor: a structural mosaic of hematopoietin receptor, immunoglobulin, and fibronectin domains.

We report the isolation from a placental library, of two cDNAs that can encode high affinity receptors for granulocyte colony-stimulating factor (G-CSF) when expressed in COS-7 cells. The cDNAs are predicted to encode integral membrane proteins of 759 and 812 amino acids in length. The predicted extracellular and membrane spanning sequences of the two clones are identical, as are the first 96 amino acids of their respective cytoplasmic regions. Different COOH termini of 34 or 87 residues are predicted for the two cDNAs, due apparently to alternate splicing. The receptor with the longer cytoplasmic domain is the closest human homologue of the murine G-CSF receptor recently described by Fukunaga et al. (Fukunaga, R., E. Ishizaka-Ikeda, Y. Seto, and S. Nagata. 1990. Cell. 61:341). A hybridization probe derived from the placental G-CSF receptor cDNA detects a approximately 3-kb transcript in RNAs isolated from placenta and a number of lymphoid and myeloid cells. The extracellular region of the G-CSF receptors is composed of four distinct types of structural domains, previously recognized in other cell surface proteins. In addition to the two domains of the HP receptor family-defining region (Patthy, L. 1990. Cell. 61:13) it incorporates one NH2-terminal Ig-like domain, and three additional repeats of fibronectin type III-like domains. The presence of both an NH2-terminal Ig-like domain and multiple membrane-proximal FN3-like domains suggests that the G-CSF receptor may be derived from an ancestral NCAM-like molecule and that the G-CSF receptor may function in some adhesion or recognition events at the cell surface in addition to the binding of G-CSF.

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.

LERK-2, a binding protein for the receptor-tyrosine kinase ELK, is evolutionarily conserved and expressed in a developmentally regulated pattern.

We have isolated and characterized cDNA clones that encode the rat homologue of a binding protein, LERK-2, for the receptor tyrosine kinase, elk. The cDNAs contain an open reading frame of 1527 nucleotides capable of encoding a protein 345 amino acid residues in length. The nucleotide sequence of the present clones is > 90% identical to the previously identified human LERK-2 cDNA, and the predicted proteins encoded by the rat and human clones are identical at 95% of amino acid residues. Recombinant proteins expressed from the rat cDNAs bind to elk with high affinity, similar to recombinant human LERK-2 and an endogenously-expressed rat elk-binding protein. Expression of the rat LERK-2 mRNA was detected in embryonic brain, kidney, lung, skeletal muscle, thymus, liver, and heart, and diminished in the early post-natal period. Significant LERK-2 mRNA expression in the young adult rat was restricted to the lung, kidney, heart and testes.

Pelvic imaging with a selenium-based detector (Thoravision). Comparison with screen-film radiography in 75 patients.

OBJECTIVE: To evaluate whether the selenium detector (Thoravision) provides sufficient diagnostic confidence in digital pelvic imaging compared with a conventional screen-film combination. METHODS: In 75 patients, pelvic imaging with conventional screen-film and isodose selenium radiography using a dedicated postprocessing mode was compared independently by three radiologists. The depiction of cortical and cancellous bone was evaluated in the iliac wings, sacral and pubic bones, acetabulum, femoral head, and trochanter. Demarcation of soft tissue was assessed in the iliac and trochanteric region. RESULTS: Visualization of cortical bone and soft tissue in the iliac area as well as soft tissue and cortical and cancellous bone in the trochanteric region was significantly superior with the selenium detector. However, conventional imaging was better in the trabecular bone of the sacral region, where results with the selenium system were particularly poor. CONCLUSIONS: The selenium detector (Thoravision) is advantageous in imaging soft tissue adjacent to the iliac wings and the trochanter, but results for the cancellous sacral bone are poor. Further modifications of postprocessing modes may lead to improved depiction of this critical pelvic area.

[The value of a new digital laser card for image/report documentation and transmission]. / Wertigkeit einer neuen digitalen Laserkarte zur Bild-/Befunddokumentation und Ubermittlung.

The capabilities of a patient-oriented digital optical laser-card for the documentation of the image/report unit and for image transmission were assessed. 150 conventional X-rays covering the fields of urology (n = 50), traumatology (n = 50) and orthopaedics (n = 50) were digitised using a CCD scanner and subsequently transmitted to an Image-Transfer Medium (ITM) and to an optical laser-card. The image quality for the detection of relevant diagnostic parameters was evaluated by 4 radiologists and one clinician of the corresponding specialty. Based upon a total of 4740 decision readings for each method, it was found that the optical laser-card reduced the image quality significantly (p < 0.01) in comparison to the digitised ITM images in all fields. Thus, a primary diagnostic statement cannot be made based upon the images of the optical card. However, concomitant documentation of the image and opinion on the card may be used for the transmission of the radiological report, especially to external referring institutions.

Ultrastructural features of alveolar cells in experimental hypersensitivity pneumonitis.

An experimental model of hypersensitivity pneumonitis in rats was used to analyze ultrastructural changes in cellular elements of the epithelium and alveolar lumen, in an attempt to correlate the immunological mechanisms responsible for these pulmonary lesions. Semifine and ultrafine sections of pulmonary tissue of immunized and intratracheally challenged rats were analyzed and compared with their respective controls. A thickening of the alveolar walls and an increase in the number of macrophages and type II pneumocytes were observed in the semifine sections. The ultrastructural examination revealed appreciable changes in morphology, size and location of both types of cells. The membranes of the macrophages showed evident alterations and the type II pneumocytes, an increase in size and number of cytoplasmic inclusions corresponding to surfactant. The cellular changes observed are consistent with phenomena of cellular activation, which can be attributed to the release of soluble mediators by T lymphocytes. The important delayed hypersensitivity phenomena based on the morphology of pulmonary lesions in this model contribute data to the pathogenic interpretation of hypersensitivity alveolitis.

Characterization of the human and murine IL-7 genes.

IL-7 cDNA clones were used to isolate clones from the human IL-7 gene locus. Characterization of the clones revealed that the human IL-7 gene contains six exons, distributed over more than 33-kbp. An 18 amino acid insert found in human IL-7, for which no counterpart has yet been demonstrated in murine IL-7, is exactly encoded by exon 5 of the human gene. Clones were also isolated containing 5' flanking sequences and the first four exons of the murine IL-7 gene. RNase protection studies of murine IL-7 mRNA, as well as the sequences of 5'-terminal murine IL-7 cDNA clones obtained by anchored polymerase chain reaction cloning, indicate that the murine IL-7 gene initiates transcription at multiple sites within a 200-bp region. This region, and the sequence upstream of this region, appears to lack transcriptional regulatory sequences commonly found in eukaryotic promoters, including the TATA and CAAT sequences. However, the region lies within a CpG island, and contains potential recognition sequences for the "helix-loop-helix" class of DNA binding proteins.

Alternate splicing of mRNAs encoding human mast cell growth factor and localization of the gene to chromosome 12q22-q24.

Human mast cell growth factor (MGF) complementary DNAs (cDNAs) were cloned from HeLa cells using the polymerase chain reaction with oligonucleotides corresponding to murine and human MGF sequences. Sequencing of the cloned human MGF polymerase chain reaction products revealed two types of cDNA: a full length form corresponding in size to the murine cDNA, and an alternately spliced clone with a deletion of the sixth exon of the gene. Since membrane-bound MGF is predicted to be proteolytically cleaved within the sequences encoded by exon 6 to generate a soluble protein, this alternately spliced cDNA would likely encode a noncleavable, membrane-bound form of MGF. No difference in biological activity on human bone marrow cells was observed with recombinant, soluble forms of both types of human MGF protein. Our previous localization of the murine MGF gene to the Sl locus on chromosome 10 suggested (via conserved linkage groups) that the human MGF gene would be located on human chromosome 12. Therefore, rodent-human somatic cell hybrids with or without an entire human chromosome 12 and hybrids retaining partial 12 were tested by Southern blot analysis and used to show the presence of the human Mgf locus at chromosome region 12q. Chromosomal in situ hybridization localized the gene to 12q22-q24 in the region predicted by the comparative mapping of the murine Mgf/Sl locus.

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.

Cloning of the human and murine interleukin-7 receptors: demonstration of a soluble form and homology to a new receptor superfamily.

cDNA clones encoding the human and murine interleukin-7 (IL-7) receptor were isolated and expressed in COS-7 cells. Binding of radiolabeled IL-7 to the recombinant IL-7 receptors produced curvilinear Scatchard plots containing high and low affinity classes. These binding properties, as well as the molecular size of the cloned receptor, were comparable to the native forms of the IL-7 receptor. In addition, several cDNA clones were isolated that encode a secreted form of the human IL-7 receptor capable of binding IL-7 in solution. Analysis of the sequence of the IL-7 receptor revealed significant homology in the extracellular domain to several recently cloned cytokine receptors, demonstrating that the IL-7 receptor is a member of a new receptor superfamily.

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.