Magnetic sorting of membrane associated IgG for phenotype-based selection of stable antibody producing cells.

To establish a simple and widely accessible technique for rapidly selecting high producing Chinese hamster ovary (CHO) cells engineered to express a monoclonal antibody (mAb), we have exploited the transient display of recombinant protein on their cell surface. In combination with magnetic bead-based methods, we demonstrate the ability to select for cells of high productivity in the absence of any metabolic-based selection method. This technique is sufficient to obtain genetically stable engineered CHO cells via a single step of cell subcloning and yields sought-after stable, high IgG producing clonal cell lines. This technique may also be applied to other types of cells as well as polyclonal Ab cell pools.

Induction of murine adenosine A(2A) receptor expression by LPS: analysis of the 5' upstream promoter.

Non-activated macrophages express low levels of A(2A)Rs and lipopolysaccharides (LPS) upregulates A(2A)R expression in an NF-κB-dependent manner. The murine A(2A)R gene is encoded by three exons, m1, m2 and m3. Exons m2 and m3 are conserved, while m1 encodes the 5' untranslated UTR. Three m1 variants have been defined, m1A, m1B and m1C, with m1C being farthest from the transcriptional start site. LPS upregulates A(2A)Rs in primary murine peritoneal and bone-marrow-derived macrophages and RAW264.7 cells by selectively splicing m1C to m2, through a promoter located upstream of m1C. We have cloned ∼1.6 kb upstream of m1C into pGL4.16(luc2CP/Hygro) promoterless vector. This construct in RAW 264.7 cells responds to LPS, and adenosine receptor agonists augmented LPS responsiveness. The NF-κB inhibitors BAY-11 and triptolide inhibited LPS-dependent induction. Deletion of a key proximal NF-κB site (402-417) abrogated LPS responsiveness, while deletion of distal NF-κB and C/EBPß sites did not. Site-directed mutagenesis of CREB (309-320), STAT1 (526-531) and AP2 (566-569) sites had little effect on LPS and adenosine receptor agonist responsiveness; however, mutation of a second STAT1 site (582-588) abrogated this responsiveness. Further analysis of this promoter should provide valuable insights into regulation of A(2A)R expression in macrophages in response to inflammatory stimuli.

The ciliary neurotrophic factor receptor alpha component induces the secretion of and is required for functional responses to cardiotrophin-like cytokine.

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.

Signaling pathways recruited by the cardiotrophin-like cytokine/cytokine-like factor-1 composite cytokine: specific requirement of the membrane-bound form of ciliary neurotrophic factor receptor alpha component.

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.

CLF associates with CLC to form a functional heteromeric ligand for the CNTF receptor complex.

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.

Cloning of MMP-26. A novel matrilysin-like proteinase.

A cDNA encoding a novel human matrix metalloproteinase (MMP), named MMP-26, was cloned from fetal cDNA. The deduced 261-amino-acid sequence is homologous to macrophage metalloelastase (51.8% identity). It includes only the minimal characteristic features of the MMP family: a signal peptide, a prodomain and a catalytic domain. As with MMP-7, this new MMP does not comprise the hemopexin domain, which is believed to be involved in substrate recognition. A study of MMP-26 mRNA steady states levels reveals, among the tissue examined, a specific expression in placenta. MMP-26 mRNA could also be detected in several human cell lines such as HEK 293 kidney cells and HFB1 lymphoma cells. Recombinant MMP-26 was produced in mammalian cells and used to demonstrate a proteolytic activity of the enzyme on gelatin and beta-casein.

BSMAP, a novel protein expressed specifically in the brain whose gene is localized on chromosome 19p12.

Using the sequence of cosmids derived from chromosome 19p12, we have identified a gene encoding a novel protein, BSMAP (brain-specific membrane-anchored protein) and cloned cDNA encoding the full-length open reading frame. Northern blot analysis revealed that BSMAP mRNA is preferentially expressed at a high level in the brain. BSMAP has a putative transmembrane domain and is predicted to be a type-I membrane glycoprotein. Genomic sequence analysis revealed that the gene encoding BSMAP consists of eight exons spanning approximately 8 kb and lies 6 kb away from the gene encoding CLF-1 in a reverse orientation. Although no candidate genetic disorders were found to map either to this precise region of chromosome 19 or to the syntenic region of the mouse genome, the highly specific expression of BSMAP mRNA suggests a role for the protein in CNS function.

A soluble form of IL-13 receptor alpha 1 promotes IgG2a and IgG2b production by murine germinal center B cells.

A functional IL-13R involves at least two cell surface proteins, the IL-13R alpha 1 and IL-4R alpha. Using a soluble form of the murine IL-13R alpha 1 (sIL-13R), we reveal several novel features of this system. The sIL-13R promotes proliferation and augmentation of Ag-specific IgM, IgG2a, and IgG2b production by murine germinal center (GC) B cells in vitro. These effects were enhanced by CD40 signaling and were not inhibited by an anti-IL4R alpha mAb, a result suggesting other ligands. In GC cell cultures, sIL-13R also promoted IL-6 production, and interestingly, sIL-13R-induced IgG2a and IgG2b augmentation was absent in GC cells isolated from IL-6-deficient mice. Furthermore, the effects of the sIL-13R molecule were inhibited in the presence of an anti-IL-13 mAb, and preincubation of GC cells with IL-13 enhanced the sIL-13R-mediated effects. When sIL-13R was injected into mice, it served as an adjuvant-promoting production to varying degrees of IgM and IgG isotypes. We thus propose that IL-13R alpha 1 is a molecule involved in B cell differentiation, using a mechanism that may involve regulation of IL-6-responsive elements. Taken together, our data reveal previously unknown activities as well as suggest that the ligand for the sIL-13R might be a component of the IL-13R complex or a counterstructure yet to be defined.

Identification of three alternatively spliced variants of human CD28 mRNA.

CD28, expressed by T cells, plays a central role in providing costimulatory signals to T cells. The cd28 gene is organized into 4 exons. An alternatively spliced CD28 mRNA lacking most of the exon 2 has been previously evidenced. We report here that non stimulated human T cells express three additional alternatively spliced variants of CD28 mRNA (CD28a-c) in. The CD28a variant, expressed at similar levels to that of the full length CD28 mRNA encoding for the membrane form, lacks exon 3. This deletion introduces (i) a frame shift resulting in the addition of two extra amino acids and a premature stop codon and, (ii) induces the loss of the transmembrane region, suggesting that it could encodes for a soluble monomeric molecule which conserves the binding sites of CD28. The CD28b and CD28c variants, expressed at a low level compared with CD28a, are generated by deletion of most of the 3' end of exon 2 plus exon 3 and exon 2 plus exon 3, respectively. Activated T cells express only the membrane CD28 mRNA. These results suggest that resting human T cells may constitutively express both membrane and soluble CD28 which can differentially regulate the outcome of the T cell response.

The distribution of IL-13 receptor alpha1 expression on B cells, T cells and monocytes and its regulation by IL-13 and IL-4.

To study the expression of IL-13 receptor alpha1 (IL-13Ralpha1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL-13Ralpha1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38- B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD-CD38- B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up-regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL-13Ralpha1 expression levels on monocytes. While IL-13Ralpha1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL-13Ralpha1 might have functions unrelated to the capacity to form a type II IL-4/IL-13R with IL-4Ralpha.

Cytokine-like factor-1, a novel soluble protein, shares homology with members of the cytokine type I receptor family.

In this report we describe the identification, cloning, and expression pattern of human cytokine-like factor 1 (hCLF-1) and the identification and cloning of its murine homologue. They were identified from expressed sequence tags using amino acid sequences from conserved regions of the cytokine type I receptor family. Human CLF-1 and murine CLF-1 shared 96% amino acid identity and significant homology with many cytokine type I receptors. CLF-1 is a secreted protein, suggesting that it is either a soluble subunit within a cytokine receptor complex, like the soluble form of the IL-6R alpha-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The highest levels of hCLF-1 mRNA were observed in lymph node, spleen, thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with constitutive expression of CLF-1 mRNA detected in a human kidney fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA was up-regulated by TNF-alpha, IL-6, and IFN-gamma. Western blot analysis of recombinant forms of hCLF-1 showed that the protein has the tendency to form covalently linked di- and tetramers. These results suggest that CLF-1 is a novel soluble cytokine receptor subunit or part of a novel cytokine complex, possibly playing a regulatory role in the immune system and during fetal development.

Production of vascular endothelial growth factor by murine macrophages: regulation by hypoxia, lactate, and the inducible nitric oxide synthase pathway.

Murine thioglycolate-induced peritoneal macrophages (MPMs) and the murine RAW264.7 macrophage-like cell line (RAW cells) constitutively produce vascular endothelial growth factor (VEGF). VEGF production is increased under hypoxic conditions or after cell activation with interferon-gamma (IFNgamma) and endotoxin (lipopolysaccharide, LPS). In contrast, tumor necrosis factor-alpha is produced only by IFNgamma/LPS-activated cells. Lactate (25 mmol/L) does not increase VEGF production by these cells. However, hypoxia, lactate, and IFNgamma/LPS-activated MPMs express angiogenic activity, whereas normoxic, nonactivated MPMs do not. Lack of angiogenic activity is not due to an antiangiogenic factor(s) in the medium of these cells. Angiogenic activity produced by hypoxia and lactate-treated MPMs is neutralized by anti-VEGF antibody, which also neutralizes most of the angiogenic activity produced by IFNgamma/LPS-activated MPMs. The inducible nitric oxide synthase inhibitors Ng-nitro-L-arginine-methyl ester (1.5 mmol/L) and aminoguanidine (1 mmol/L) block production of angiogenic activity by MPMs and RAW cells. In RAW cells, Ng-nitro-L-arginine-methyl ester and AG block IFNgamma/LPS-activated, but not constitutive, VEGF production, whereas in MPMs, neither constitutive nor IFNgamma/LPS-activated VEGF synthesis is affected. Synthesis of tumor necrosis factor-alpha is also unaffected. In contrast to normoxic, nonactivated MPMs, inducible nitric oxide synthase-inhibited, IFNgamma/LPS-activated MPMs produce an antiangiogenic factor(s). We conclude that VEGF is a major contributor to macrophage-derived angiogenic activity, and that activation by hypoxia, lactate, or IFNgamma/LPS switches macrophage-derived VEGF from a nonangiogenic to an angiogenic state. This switch may involve a posttranslational modification of VEGF, possibly by the process of ADP-ribosylation. ADP-ribosylation by MPM cytosolic extracts or by cholera toxin switches rVEGF165 from an angiogenic to a nonangiogenic state. In IFNgamma/LPS-activated MPMs, the inducible nitric oxide synthase-dependent pathway also regulates the expression of an antiangiogenic factor(s) that antagonizes the bioactivity of VEGF and provides an additional regulatory pathway controlling the angiogenic phenotype of macrophages.

A novel 4-kb interleukin-13 receptor alpha mRNA expressed in human B, T, and endothelial cells encoding an alternate type-II interleukin-4/interleukin-13 receptor.

A 4 kb human interleukin-13 receptor (IL-13R) chain cDNA was cloned from a B cell cDNA library using expressed sequence tags homologous to mouse IL-13R as probes. The deduced protein sequence shows a significant level of sequence identity with the IL-5R and the human IL-13R identified recently by expression cloning. The cytoplasmic region is very highly conserved between human and mouse homologs and contains a consensus binding motif for a signal transducer and activator of transcription. The cDNA encodes a protein binding IL-13 when expressed alone which participates in a receptor complex for both IL-4 and IL-13 when expressed in conjunction with the IL-4R alpha chain. Transcripts for this IL-13R chain could be detected in most tissues and organs studied and in T, B, endothelial cells, basophilic, immature mast cell, and monocytic cell lines. The pattern of expression is different from the other recently cloned IL-13R molecule, and correlates with sites where IL-4 and IL-13 signaling is known to occur. This novel receptor is therefore likely to be implicated in reactions involved in IgE responses, T helper 2 differentiation, adhesion of leukocytes to endothelium, and therefore in pathological phenomena such as allergy, atopy, and asthma.

Acetyl-CoA carboxylase gene expression in the developing mouse brain. Comparison with other genes involved in lipid biosynthesis.

The present study documents the steady-state levels for the mRNAs encoding acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD2) and brain long-chain acyl-CoA synthetase (BLACS) during mouse brain development. It is shown that ACC and FAS mRNA levels are at a maximum 5 days after birth, a time when cell proliferation is intense in the mouse brain, and then decrease steadily to reach 20% of those maximal values at day 20. The ACC transcript isoforms, which were detected in the central nervous system (CNS), originated from promoter P2 of the ACC gene. They encode ACC enzymes which cannot be phosphorylated at the Ser-1200 locus, thus indicating that brain ACC is highly sensitive to citrate activation. The developmental pattern for the SCD2 mRNA level is different from that of true myelin genes, such as CGT. Indeed, the steady-state levels for SCD2 and CGT in 5-day-old brain represent 85% and 5% of their maximal values, respectively. BLACS expression rose during the developmental period studied, but a slow decrease in the mRNA levels was not observed after postnatal day 20, unlike in "myelin-specific' genes. Therefore, it appears that the expression of the genes involved in fatty acid biosynthesis is independent of the myelinating signal in the mouse CNS.

Human native soluble CD40L is a biologically active trimer, processed inside microsomes.

CD40 ligand (CD40L) is a glycoprotein expressed on the surface of activated helper T cells, basophils, mast cells, and eosinophils. Binding of CD40L to its receptor CD40 on the B cell surface induces B cell proliferation, adhesion, and immunoglobulin class switching. We have identified soluble cleavage products of human CD40L in the supernatant of a stimulated human T cell clone. Subcellular fractionation experiments have shown that the transmembrane CD40L is processed inside the microsomes and that its cleavage is stimulation-dependent. The native human soluble CD40L is trimeric and, when used in conjunction with interleukin-4, induces B cell proliferation.

Cleavage of membrane-bound CD40 ligand is not required for inducing B cell proliferation and differentiation.

The physical interaction between the B cell surface molecule CD40 and its ligand, CD40L, is known to be crucial in the development and maintenance of humoral immunity. Recently it has been shown that the CD40L is processed and that its soluble cleavage products are released into the extracellular environment. To study the functions of soluble and membrane-bound human CD40L on human B cells, we generated an uncleavable CD40L cDNA deletion mutant. The activities of transfectants expressing either mutated or wild-type CD40L were then compared on human B cells. Both the soluble and the uncleavable membrane-bound CD40L were able to induce, in conjunction with interleukin-4, B cell proliferation and IgE synthesis. Therefore, membrane-bound and soluble CD40L exhibit the same pattern of activities on B cells and membrane CD40L cleavage is not a prerequisite for its function.

Human CD40-ligand: molecular cloning, cellular distribution and regulation of expression by factors controlling IgE production.

Here we report the cloning of the cDNA for human CD40-Ligand (CD40-L) from a CD4-positive T cell clone. The deduced amino acid sequence predicts a type II membrane protein of 261 amino acids. Northern blot and FACS analysis of PBMNC revealed that the human CD40-L can be detected on T cells and is absent from B cells and monocytes. The human CD40-L is expressed on both CD4- and CD8-positive T cells, (CD45R0+) and (CD45RA+) subsets. We observed that IL-4, an inducer of IgE production, upregulated CD40-L mRNA level while IFN gamma, an inhibitor of IgE synthesis, reduced the expression of CD40-L mRNA. These data suggest a the correlation between human CD40-L expression and IgE production.