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Biomedical subjects

J Tavernier

Publications and source records attributed to J Tavernier.

At least 73 records · Page 4Linked to original sources
PubMed1993

Expression, purification and crystallization of fully active, glycosylated human interleukin-5.

Recombinant human interleukin-5 (hIL-5) has been expressed at high levels and produced in large quantities in baculovirus infected Sf9 insect cells. The glycosylated protein was purified using immuno-affinity chromatography and gel filtration. Purified hIL-5 has been crystallized using standard vapour diffusion techniques with PEG as a coprecipitant. The crystals belong to the C2 space group and diffract to 2 A.

Animals↗
PubMed1993

Recombinant soluble human interleukin-5 (hIL-5) receptor molecules. Cross-linking and stoichiometry of binding to IL-5.

Recombinant soluble human interleukin-5 receptor alpha (shIL-5R alpha) has been expressed in COS-1 cells and in baculovirus-infected cells. The protein was purified from the supernatant by chromatography on concanavalin A-Sepharose, MonoQ, and a final gel filtration step. A chimeric fusion receptor protein (hIL-5R alpha-h gamma 3) was constructed by fusion of the cDNA corresponding to the shIL-5R alpha to the cDNA corresponding to the Fc part of the human IgG C gamma 3 chain, and was expressed in baculovirus-infected insect cells. The chimeric receptor was secreted as a disulfide-linked homodimer, and was purified by protein G affinity chromatography. In a solid-phase binding assay the shIL-5R alpha and the bivalent hIL-5R alpha-h gamma 3 were found to bind hIL-5 with a similar affinity, corresponding to the membrane-bound, low affinity hIL-5R alpha. SDS-polyacrylamide gel electrophoresis of shIL-5R alpha cross-linked to radiolabeled hIL-5, suggested that one shIL-5R alpha molecule binds to one hIL-5 homodimer molecule. Gel filtration studies of the complex formed between the shIL-5R alpha and hIL-5 pointed toward the same stoichiometry of binding. The formation of such a complex could be observed by electrophoresis in native gels. Immunoaffinity chromatography using a non-neutralizing monoclonal antibody directed against hIL-5, followed by size column chromatography, allowed the purification of the complex. The data obtained from the amino acid analysis of the constituents of the complex blotted from an SDS-polyacrylamide gel, and from the amino acid composition of the complex blotted from a native polyacrylamide gel, provided direct evidence that the shIL-5R alpha binds the hIL-5 dimer in a 1:1 ratio.

Animals↗
PubMed1993

Human TNF mutants with selective activity on the p55 receptor.

The remarkable ability of tumour necrosis factor (TNF), especially in combination with interferon, selectively to kill or inhibit malignant cell lines is so far unmatched by any other combination of cytokines. But clinical trials in cancer patients have on the whole been disappointing, and it has been estimated that a TNF dose would be effective only at 5-25 times the maximum tolerated dose. High TNF concentrations give a much more pronounced antitumour activity in mice, in which murine TNF is about 50-fold more systemically toxic than human TNF. But there is little or no species specificity in cytotoxicity of murine TNF and human TNF on human as well as on murine cell lines. This dual action of TNF may be explained by the existence of two types of receptor for TNF: the smaller, TNF-R55, is present on most cells and particularly on those susceptible to the cytotoxic action of TNF; the larger, TNF-R75, is also present on many cell types, especially those of myeloid origin, and is strongly expressed on stimulated T and B lymphocytes. In mice, human TNF binds only to murine TNF-R55 (ref. 15), which can then mediate cytotoxic activity on malignant cells. As human TNF does not bind to murine TNF-R75, the latter must be responsible for the much enhanced systemic toxicity of murine TNF. Human TNF can, however, become toxic in mice when a second pathway is activated. There is no reciprocal situation in the human system: human and murine TNF bind almost equally well to the two human TNF receptors. Here we describe human TNF mutants that sill interact with the human TNF-R55 receptor but which have largely lost their ability to bind to human TNF-R75. Activation of TNF-R55 is sufficient to trigger cytotoxic activity towards transformed cells. One representative human TNF mutant retains its antitumour activity in nude mice carrying tumours derived from human cancers. Under the appropriate conditions, such human TNF mutants are expected to induce less systemic toxicity in man, while still exerting their direct antitumour effect.

Adenocarcinoma↗
PubMed1992

Molecular basis of the membrane-anchored and two soluble isoforms of the human interleukin 5 receptor alpha subunit.

By use of a 3' extension PCR strategy, cDNA clones were isolated spanning the transmembrane region and a complete cytoplasmic domain of the human interleukin 5 receptor alpha subunit (hIL5R alpha). These cDNAs differ from previously isolated clones encoding a soluble hIL5R alpha form by a sequence switch at position 1243. When expressed in COS-1 cells, only low-affinity binding of 125I-labeled human interleukin 5 was observed. Coexpression of the hIL5R beta chain led to a 2-fold increase in binding affinity. In addition, this same cloning strategy allowed us to identify a putative second soluble isoform of hIL5R alpha. Genomic data revealed that the two soluble variants arise from either a "normal" splicing event or from the absence of splicing, whereas synthesis of the membrane-anchored form requires alternative splicing.

Amino Acid Sequence↗
PubMed1992

Tumor necrosis factor causes bronchial hyperresponsiveness in rats.

We have previously reported that exposure of rats to aerosolized endotoxin (LPS) causes a transient, dose-dependent increase in bronchial responsiveness (BR) to 5 hydroxytryptamine (5HT), 90 min after exposure. In the present study we examined whether LPS induces the release of tumor necrosis factor (TNF) in the airways and whether TNF contributes to the increase in BR. After 90 min following exposure to aerosolized LPS, at a concentration of 1 or 10 micrograms/ml, TNF concentrations in bronchoalveolar lavage (BAL) fluid were 17.9 +/- 6.9 and 80.5 +/- 7.8 U/ml, respectively. No TNF was detected in BAL fluid of saline-exposed animals. At 90 min after exposure to aerosolized recombinant human TNF (rhTNF) (1 microgram/ml) an increase in BR was observed: the provocative concentration of 5HT causing a 50% increase in lung resistance (PC50RL 5HT) was 2.7 +/- 0.4 versus 4.4 +/- 0.3 microgram/kg in saline-exposed animals (p less than 0.01). Pretreatment with anti-TNF antibodies 30 min before LPS exposure significantly diminished the increase in BR: PC50RL 5HT was 2.3 +/- 0.4 versus 1.2 +/- 0.5 microgram/kg in control pretreated LPS-exposed rats (p less than 0.01). Exposure to aerosolized TNF also induced a significant influx of neutrophils in BAL fluid (12.1 +/- 3.7 versus 1.7 +/- 0.4% in saline-exposed animals) (p less than 0.01). The LPS-induced neutrophil influx in BAL fluid was partly inhibited by pretreatment with anti-TNF antibodies (55.2 +/- 5.1 versus 76.0 +/- 3.9%) (p less than 0.01). We conclude that TNF causes bronchial hyperresponsiveness and airway inflammation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aerosols↗
PubMed1991

Characterization of the murine IL-5 receptor complex with the use of a panel of monoclonal antibodies. Relationship to the murine IL-3 receptor.

To obtain mAb against the murine IL-5R (mIL-5R), Wistar rats were immunized with B13 cells, a murine Ly-1+ (CD5+) pre-B cell line which is dependent on IL-3 or IL-5 for its growth. A first group of six mAb could immunoprecipitate, from detergent-lysed B13 cells, a 60-kDa polypeptide (p60) corresponding to the recently cloned mIL-5R alpha-chain. A second group of three mAb was able to immunoprecipitate a protein doublet of 130 to 140 kDa (p130 and p140) corresponding to the previously characterized mIL-3R and mIL-3R-like polypeptide, respectively. One mAb (25C9) specifically bound the p130 polypeptide only. Here we show that: 1) mAb directed against the mIL-5R p60 component completely block IL-5 binding; 2) mAb recognizing the p130-p140 doublet interfere with both IL-3 and IL-5 binding; 3) mAb recognizing p130-p140 block the high affinity binding of IL-5 and hence the high affinity mIL-5R consists of the association of the p60 and p130 and/or p140 component; 4) one particular mAb, 25C9, which binds only to the p130 polypeptide, interferes with only IL-3 binding, and has no effect on the binding of IL-5. These results on binding were corroborated by a biologic assay based on the cytokine-dependent proliferation of B13 cells. The results presented here support a model for the mIL-5R consisting of the alpha-chain (p60) associated with the p140 (IL-3R-like), whereas the p130 (IL-3R) is not involved in the IL-5R complex.

Animals↗
PubMed1991

A human high affinity interleukin-5 receptor (IL5R) is composed of an IL5-specific alpha chain and a beta chain shared with the receptor for GM-CSF.

cDNA clones encoding two receptor proteins involved in the binding of human interleukin 5 (hIL5) have been isolated. A first class codes for an IL5-specific chain (hIL5R alpha). The major transcript of this receptor gene, as analyzed in both HL-60 eosinophilic cells and eosinophilic myelocytes grown from cord blood, encodes a secreted form of this receptor. This soluble hIL5R alpha has antagonistic properties. A second component of the hIL5R is found to be identical to the beta chain of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) high affinity receptor. The finding that IL5 and GM-CSF share a receptor subunit provides a molecular basis for the observation that these cytokines can partially interfere with each other's binding and have highly overlapping biological activities on eosinophils.

Amino Acid Sequence↗
PubMed1991

Amino acid sequence analysis of a mouse interleukin 5 receptor protein reveals homology with a mouse interleukin 3 receptor protein.

A polypeptide chain for the mouse interleukin 5 receptor (IL5R) was purified from detergent-lysed B13 cells, a mouse IL5-dependent pre-B cell line. Purification was by a single immunoaffinity chromatographic step using an anti-mouse IL5R monoclonal antibody, R52. Internal amino acid sequence was obtained from four trypsin-generated peptides. All peptides were found to be present in the published amino acid sequence of a mouse IL3R and the mouse IL3R-like protein deduced from the cDNA. This indicates that the mouse IL5R and the mouse IL3R have a homologous polypeptide in common and suggests that the specificity of these lymphokine receptors is mainly generated by association with another ligand-specific polypeptide chain.

Amino Acid Sequence↗
PubMed1991

Molecular basis of a high affinity murine interleukin-5 receptor.

The mouse interleukin-5 receptor (mIL-5R) consists of two components one of which, the mIL-5R alpha-chain, binds mIL-5 with low affinity. Recently we demonstrated that monoclonal antibodies (Mabs) recognizing the second mIL-5R beta-chain, immunoprecipitate a p130-140 protein doublet which corresponds to the mIL-3R and the mIL-3R-like protein, the latter chain for which so far no ligand has been identified. In this study we show that a high affinity mIL-5R can be reconstituted on COS1 cells by co-expression of the mIL-5R alpha-chain with the mIL-3R-like protein (beta-chain). Cross-linking of 125I-labeled mIL-5 to the COS1 cells co-transfected with both cDNAs revealed the same pattern as in B13 cells, i.e. two proteins of 60 and 130 kd which correspond to the low affinity mIL-5R alpha-chain and the mIL-3R-like protein, respectively. The dissociation rate of mIL-5 from this reconstituted high affinity site was lower than that of the low affinity site, whereas the association rate was unchanged. Nonetheless, the apparent dissociation constant (Kd) for this reconstituted receptor was still 10-fold higher than the Kd observed for B13 cells. Although the mIL-3R is greater than 90% homologous to the mIL-3R-like protein, no increase in affinity for mIL-5 was detected on COS1 cells co-transfected with the cDNAs for the mIL-5R alpha-chain and the mIL-3R protein.

Animals↗
PubMed1991

Localization of the active site of human tumour necrosis factor (hTNF) by mutational analysis.

In order to define the active site(s) of human tumour necrosis factor (hTNF), we mutagenized its gene at random and directly screened the resulting population for loss of cytotoxic activity on L929 cells. Four biologically inactive mutant proteins (Arg32----Trp, Leu36----Phe, Ser86----Phe and Ala84----Val) behaved similar to the wild-type in various physico-chemical assays. The residues were positioned on a 3D structural model and were found to cluster together at the base of the molecule at each side of the groove that separates two monomers in the trimeric structure. A very conservative mutation at one of these sites (Ala84----Val) almost completely abolished cytotoxic activity. Amino acid alterations in three other residues in close proximity to this receptor binding site were introduced: replacements at positions 29 and 146 clearly reduced cytotoxicity only when non-conservative alterations were introduced (Leu29----Ser and Glu146----Lys), suggesting an indirect influence on the active site. However, a conservative mutation at position 91 (Val----Ala) caused a significant drop (500-fold) in bioactivity which suggests that Val91 may also play a direct role in receptor recognition. Our results favor a model in which each TNF molecule has three receptor-interaction sites (between the three subunits), thus allowing signal transmission by receptor clustering.

Amino Acid Sequence↗
PubMed1990

Characterization of a receptor for interleukin 5 on human eosinophils: variable expression and induction by granulocyte/macrophage colony-stimulating factor.

Interleukin 5 (IL-5) acts on eosinophil differentiation and activation, suggesting the existence of a membrane receptor for IL-5 on eosinophils. Here, we report that 125I-labeled recombinant human IL-5 bound, at 4 degrees C, to high affinity receptors on human eosinophils. The association constant was higher for hypodense eosinophils (1.93 x 10(9) M-1) than for normodense cells (0.39 x 10(9) M-1), with a closely related number of receptor sites per cell. No specific binding occurred on neutrophils. The specific binding of IL-5 was induced by overnight incubation at 37 degrees C of human eosinophils with granulocyte/macrophage (GM)-CSF. The levels of increase were significantly higher for normodense than for hypodense eosinophils, suggesting a previous in vivo activation of the later subpopulation by GM-CSF. IL-3 was ineffective by itself but synergistically enhanced the effect of GM-CSF. Specificity studies showed that the binding of 125I-labeled IL-5 was inhibited by IL-5, but not by other cytokines, on human eosinophils. These results show the existence of a specific binding site for IL-5 on human eosinophils with a variable affinity on eosinophil hypodense or normodense subpopulations, as previously reported for other membrane receptors.

Cells, Cultured↗
PubMed1990

Expression of the murine interleukin-5 receptor on Xenopus laevis oocytes.

In this study we describe the use of Xenopus laevis oocytes for the detection of mRNA coding for a murine interleukin-5 (mI15) receptor. When injected with sucrose gradient fractionated polyA+ RNA derived from the murine 115-dependent pre B cell line B13, these oocytes could specifically bind 35S-methionine labeled mI15. A size of approximately 4000 nucleotides (25S) was estimated for the mRNA corresponding to the mIL5-binding activity. This binding was not blocked by a monoclonal antibody R52 specific for the MI15-receptor, suggesting that the oocytes express a different form of this receptor.

Animals↗
PubMed1990

Characterization of interleukin 5 receptors on eosinophilic sublines from human promyelocytic leukemia (HL-60) cells.

The T cell product interleukin 5 (IL-5) has been shown to be a key factor in the development and the maturation of the eosinophilic cell lineage. We report here on the detection of human IL-5 receptors on eosinophilic sublines of the promyelocytic leukemia HL-60. Sodium butyrate, which initiates differentiation to mature eosinophils, also induces the appearance of high affinity (Kd 1-5 X 10(-11) M) IL-5 binding sites on these cells. The receptors are specific for IL-5, since binding of radiolabeled ligand can only be inhibited with homologous or murine IL-5 and not by other cytokines. We further show that the receptors are functional, since IL-5 can stimulate the proliferation of these cells. Affinity crosslinking of surface-bound 125I human IL-5 or 35S mouse IL-5 identified two membrane polypeptides of approximately 60 and approximately 130 kD to which IL-5 is closely associated. The presence of granulocyte/macrophage-colony-stimulating factor or tumor necrosis factor during butyrate induction decreased the expression of IL-5 binding sites compared with control cultures. The identification and characterization of human IL-5 receptors on HL-60 sublines should provide new insight into the role of this cytokine in eosinophil differentiation.

Butyrates↗
PubMed1990

Analysis of the structure-function relationship of tumour necrosis factor. Human/mouse chimeric TNF proteins: general properties and epitope analysis.

To analyse the structure-function relationship of tumour necrosis factor (TNF), a set of in-frame chimeric genes was constructed by coupling appropriate segments of the human and mouse TNF coding regions. Under control of the bacteriophage lambda inducible PL promoter high level expression of these chimeric genes was obtained in Escherichia coli. Although both human and mouse TNF were produced in E. coli as soluble proteins, a reduction of solubility was observed in some of the chimeric proteins. The specific activity was variable, but in some constructs comparable to human TNF, indicating that the structural conformation of these chimeric proteins resembled the human TNF structure. Neutralization analysis using two monoclonal antibodies directed against human TNF, indicated that the regions involved in the binding of these antibodies are distributed over multiple segments of the polypeptide. Further analysis by site-directed mutagenesis of one subregion allowed the identification of the Arg131 residue as involved in the binding of both neutralizing monoclonal antibodies; an Arg131----Gln replacement abolished antibody binding but did not affect the specific activity of TNF.

Amino Acid Sequence↗
PubMed1990

Comparative activity of human and murine tumor necrosis factor in toxicity and anti-infectious assays in mice.

In comparative experiments, the lethal effect of rHuTNF or rMuTNF was evaluated in animals sensitized by adrenalectomy or galactosamine treatment. No clear-cut difference was observed in the effective dose irrespective of the origin of the TNF preparation in Swiss or in C3H/HeJ mice. The latter mouse substrain known to be a low responder to LPS was used in the assays to evaluate the influence of LPS contamination in TNF-induced responses. Like LPS, TNF was shown to induce abortion in pregnant mice, and the mortality rate of foetuses was almost the same in animals challenged with either rHuTNF or rMuTNF. No species preference was apparent in the protective effect of TNF against a bacterial challenge. In adult mice subsequently infected with Klebsiella pneumoniae or Listeria monocytogenes, the survival rate was comparable in groups treated with both TNF preparations. In contrast, in young mice rHuTNF and rMuTNF were ineffective against Listeria and poorly active against Klebsiella organisms, whereas a significant effect was obtained with crude mouse serum containing TNF.

Abortifacient Agents, Nonsteroidal↗