Biomedical subjects

J Tavernier

Publications and source records attributed to J Tavernier.

At least 55 records · Page 3Linked to original sources

PubMed1997

The beta chain of the interleukin-3 receptor functionally associates with the erythropoietin receptor.

Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors share a common beta chain (beta(c)), and both cytokines enhance erythropoietin (Epo)-dependent in vitro erythropoiesis by primary hematopoietic progenitors and factor-dependent cells. These data suggest that the Epo receptor and beta(c) may functionally interact. To determine whether such interactions can be documented, we studied a murine factor-dependent cell line (Ba/F3), which endogenously expresses IL-3R. First, Ba/F3 cells were transfected with murine EpoR, making them responsive to both IL-3 and Epo. Next, the EpoR expressing cells were transfected with murine beta(c). This resulted in an enhanced sensitivity of these cells to Epo, which was especially pronounced at low Epo concentrations. Ba/F3-EpoR were then treated with antisense oligodeoxynucleotides to the murine beta. Control sense and nonsense had no effect on Epo-dependent growth, but the antisense markedly and specifically inhibited Epo-dependent growth. In contrast, the antisense did not affect beta-globin message levels (another Epo-responsive effect in these cells) detectable by Northern blot. Finally, Western blot analysis of proteins immunoprecipitated from cells expressing both receptors with antibody against beta and blotted with antibody against EpoR, or immunoprecipitated with antibody against EpoR and blotted with antibody against beta, showed that EpoR and beta coimmunoprecipitate. These data show that the beta chain functionally and physically associates with the EpoR. This suggests that these cytokine receptors exist as a large supercomplex and offers the first molecular explanation for the synergistic effects of IL-3 and GM-CSF with Epo during erythropoiesis.

Animals↗

PubMed1997

A single STAT recruitment module in a chimeric cytokine receptor complex is sufficient for STAT activation.

We established a system of receptor chimeras that enabled us to induce heterodimerization of different cytoplasmic tails. Fusion constructs were created that are composed of the extracellular parts of the interleukin-5 receptor alpha and beta chains, respectively, and the transmembrane and intracellular parts of gp130, the signal transducing chain of the interleukin-6 receptor complex. In COS-7 transfectants we observed a dose-dependent interleukin-5-inducible STAT1 activation for which the presence of both the alpha and the beta chain chimera was needed. No STAT activity was detected if one of the cytoplasmic tails of the receptor complex was deleted, indicating that STAT activity resulted from a receptor dimer rather than from higher receptor aggregates. We further investigated whether dimerization of STAT1 depends on the juxtaposition of two STAT recruitment modules in a receptor complex. We show that a receptor dimer with only a single STAT1 docking site was still able to lead to STAT1 activation. This indicates that the formation of a paired set of STAT binding sites in a receptor complex is not the prerequisite for STAT factor dimerization. Our findings are discussed in view of alternative STAT dimerization models.

Animals↗

PubMed1997

Upregulation of alpha GM-CSF-receptor in nonatopic asthma but not in atopic asthma.

BACKGROUND: Intrinsic asthma is characterized by an increased number of activated eosinophils and macrophages and an increased expression of the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) in the bronchial mucosa. OBJECTIVE: This study was carried out to investigate the expression of alpha GM-CSF receptor (alpha GM-CSFr) messenger RNA and protein in the bronchial mucosa of patients with intrinsic or atopic asthma and of control subjects and to correlate the expression of alpha GM-CSFr to the number of EG2+ cells (eosinophils) and CD68+ cells (macrophages) and pulmonary function. METHODS: Nineteen patients with stable asthma (9 with atopic and 10 with intrinsic asthma) and 22 normal control subjects (12 atopic and 10 nonatopic subjects) were recruited, and FEV1 (percent predicted) and PC20 were measured before bronchoscopy. Endobronchial biopsy specimens were obtained and examined for membrane-bound alpha GM-CSFr by using in situ hybridization and immunocytochemistry. RESULTS: alpha GM-CSFr mRNA- and protein-positive cells were identified in biopsy specimens from all four groups studied. There was no significant difference in the number of cells expressing alpha GM-CSFr mRNA and protein in patients with atopic asthma compared with atopic and nonatopic control subjects. However, the numbers of alpha GM-CSFr mRNA- and protein-positive cells were significantly higher in nonatopic patients with asthma compared with atopic patients with asthma and atopic and nonatopic control subjects (p < 0.001). In the patients with intrinsic asthma, the number of alpha GM-CSFr mRNA-positive cells per millimeter of basement membrane correlated with numbers of CD68+ cells (r2 = 0.87, p < 0.001) but not with EG2+ cells, and colocalization studies demonstrated that 80% of the cells expressing alpha GMCSFr mRNA were CD68+. The expression of GM-CSF was also significantly increased in patients with intrinsic asthma compared with those with atopic asthma and control subjects (p < 0.05). In addition, in intrinsic asthma, there was a correlation between alpha GM-CSFr mRNA and FEV1 (r2 = 0.61, p < 0.05). CONCLUSION: These results demonstrate that elevated numbers of cells expressing alpha GM-CSFr can be detected in nonatopic asthma but not in atopic asthma and suggest that this increased expression is predominantly macrophage-associated and may play an important pathophysiologic role in intrinsic asthma.

Adult↗

PubMed1997

Membrane-bound and soluble alpha IL-5 receptor mRNA in the bronchial mucosa of atopic and nonatopic asthmatics.

Eosinophilic inflammation and interleukin-5 (IL-5) expression are characteristic features of the bronchial mucosa in asthma. We have investigated the differential expression of membrane and soluble isoforms of alpha IL-5 receptor (alpha IL-5Rm and alpha IL-5Rs) mRNA in asthmatics and in normal control subjects and examined the correlation between alpha IL-5Rm and alpha IL-5Rs expression and the FEV1 and airway hyperresponsiveness. Nineteen subjects with stable asthma (atopic = 9; intrinsic = 10) and 22 control subjects (atopic = 12; nonatopic = 10) were recruited. Endobronchial biopsies were obtained and processed for in situ hybridization and double-staining techniques. There was a significant increase in the number of cells per millimeter basement membrane expressing mRNA for total, membrane-bound, and soluble alpha IL-5R in asthmatics when compared with that in nonasthmatic control subjects (p < 0.001); 93% of the cells positive for alpha IL-5R mRNA were EG2+ve eosinophils. There was no significant difference in the expression of alpha IL-5Rm and alpha IL-5Rs between the atopic and nonatopic asthmatics. The expression of alpha IL-5Rm and alpha IL-5Rs was also nonsignificantly different between the atopic and nonatopic control subjects. However, in the asthmatic subjects, the number of positive cells expressing mRNA for alpha IL-5Rm inversely correlated with FEV1(r2 = 0.89, p < 0.001), whereas the expression of alpha IL-5Rs mRNA directly correlated with FEV1 (r2 = 0.52, p < 0.001). There were no significant correlations between alpha IL-5R isoforms and the methacholine PC20. These results suggest that alpha IL-5R upregulation and differential regulation of alternatively spliced alpha IL-5R mRNA transcripts may influence the eosinophil response and the accompanying changes in airflow limitation in both atopic and nonatopic variants of chronic asthma.

Adult↗

PubMed1996

ESM-1 is a novel human endothelial cell-specific molecule expressed in lung and regulated by cytokines.

We here report the identification of a novel human endothelial cell-specific molecule (called ESM-1) cloned from a human umbilical vein endothelial cell (HUVEC) cDNA library. Constitutive ESM-1 gene expression (as demonstrated by Northern blot and reverse transcription-polymerase chain reaction analysis) was found in HUVECs but not in the other human cell lines tested. The cDNA sequence contains an open reading frame of 552 nucleotides and a 1398-nucleotide 3'-untranslated region including several domains involved in mRNA instability and five putative polyadenylation consensus sequences. The deduced 184-amino acid sequence defines a cysteine-rich protein with a functional NH2-terminal hydrophobic signal sequence. Searches in several data bases confirmed the unique identity of this sequence. A rabbit immune serum raised against the 14-kDa COOH-terminal peptide of ESM-1 immunoprecipitated a 20-kDa protein only in ESM-1-transfected COS cells. Immunoblotting and immunoprecipitation of HUVEC lysates revealed a specific 20-kDa band corresponding to ESM-1. In addition, constitutive ESM-1 gene expression was shown to be tissue-restricted to the human lung. Southern blot analysis suggests that a single gene encodes ESM-1. A time-dependent up-regulation of ESM-1 mRNA was seen after addition of tumor necrosis factor alpha (TNFalpha) or interleukin (IL)-1beta but not with IL-4 or interferon gamma (IFNgamma) alone. In addition, when IFNgamma was combined with TNFalpha, IFNgamma inhibited the TNFalpha-induced increase of ESM-1 mRNA level. These data suggest that ESM-1 may have potent implications in the areas of vascular cell biology and human lung physiology.

Amino Acid Sequence↗

PubMed1996

Interleukin-5 receptor alpha subunit gene regulation in human eosinophil development: identification of a unique cis-element that acts lie an enhancer in regulating activity of the IL-5R alpha promoter.

Further functional and biochemical characterization of the nuclear factor(s) which interacts with the EOS1 enhancer-like element in the IL-5R alpha promoter is currently in progress. Since different transcription factors recognize and interact with DNA in distinct fashions and with distinct structural motifs, we have modeled potential binding of the EOS1 factor to its cis-element based upon its methylation interference pattern (Fig. 2), using a cylindrical DNA helical projection (Fig. 6). Over a length of two helical turns, all nuclear protein contacts indicated by methylation interference map to one side of the DNA helix, suggesting that EOS1 binds in the major groove, across the minor groove, and on only one side of the helix. Further review of the model also reveals a potential diad symmetry for the binding site, suggestive of binding by a homodimer and consistent with the formation of the two DNA-protein complexes in our electrophoretic mobility shift experiments that could represent interactions with monomer versus dimer. Comparison of the EOS1 binding motif to similar models for the binding of other transcription factor families for which structural crystallographic and/or binding data is available suggests a similarity of the EOS1 complex to that of the bacterial helix-turn-helix phage lambda and 434 repressor-operator complexes, and the Cys4 zinc finger glucocorticoid response element (GRE) DNA-binding motifs, all of which show similar diad symmetry and binding in the major groove on one side of the DNA. The possibility that EOS1 functions as a GRE is being investigated, especially since there is a consensus AP-1 site at bp -440 to -432 of the IL-5R alpha promoter, immediately adjacent to the EOS1 binding site (see Fig. 5 in reference [36]) and AP-1/GRE interactions have been identified for composite response elements in the regulation of a number of different genes. The identification or cloning of EOS1, a potentially novel and eosinophil lineage-active transcription factor, should enhance our understanding of the processes involved in eosinophil development in particular and myeloid lineage commitment and differentiation in general.

Animals↗

PubMed1995

Detailed analysis of the IL-5-IL-5R alpha interaction: characterization of crucial residues on the ligand and the receptor.

The receptor for interleukin-5 (IL-5) is composed of two different subunits. The IL-5 receptor alpha (IL-5R alpha) is required for ligand-specific binding while association with the beta-chain results in increased binding affinity. Murine IL-5 (mIL-5) has similar activity on human and murine cells, whereas human IL-5 (hIL-5) has marginal activity on murine cells. We found that the combined substitution of K84 and N108 on hIL-5 by their respective murine counterpart yields a molecule which is as potent as mIL-5 for growth stimulation of a murine cell line. Since the unidirectional species specificity is due only to the interaction with the IL-5R alpha subunit, we have used chimeric IL-5R alpha molecules to define regions of hIL-5R alpha involved in species-specific hIL-5 ligand binding. We found that this property is largely determined by the NH2-terminal module of hIL-5R alpha, and detailed analysis defined D56 and to a lesser extent E58 as important for binding. Moreover, two additional residues, D55 and Y57, were identified by alanine scanning mutagenesis within the same region. Based on the observed homology between the NH2-terminal module and the membrane proximal (WSXWS-containing) module of hIL-5R alpha we located this stretch of four amino acid residues (D55, D56, Y57 and E58) in the loop region that connects the C and D beta-strands on the proposed tertiary structure of the NH2-terminal module.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Identification of receptor-binding domains on human interleukin 5 and design of an interleukin 5-derived receptor antagonist.

A detailed structure-function analysis of human interleukin 5 (hIL5) has been performed. The hIL5 receptor is composed of two different polypeptide chains, the alpha and beta subunits. The alpha subunit alone is sufficient for ligand binding, but association with the beta subunit leads to a 2- to 3-fold increase in binding affinity. The beta chain is shared with the receptors for IL3 and granulocyte/macrophage-colony-stimulating factor--hence the descriptor beta C (C for common). All hIL5 mutants were analyzed in a solid-phase binding assay for hIL5R alpha interaction and in a proliferation assay using IL5-dependent cell lines for receptor-complex activation. Most residues affecting binding to the receptor alpha subunit were clustered in a loop connecting beta-strand 1 and helix B (mutants H38A, K39A, and H41A), in beta-strand 2 (E89A and R91A; weaker effect for E90A) and close to the C terminus (T109A, E110A, W111S, and I112A). Mutations at one position, E13 (Glu13), caused a reduced activation of the hIL5 receptor complex. In the case of E13Q, only 0.05% bioactivity was detected on a hIL5-responsive subclone of the mouse promyelocytic cell line FDC-P1. Moreover, on hIL5-responsive TF1 cells, the same mutant was completely inactive and proved to have antagonistic properties. Interactions of this mutant with both receptor subunits were nevertheless indistinguishable from those of nonmutated hIL5 by crosslinking and Scatchard plot analysis of transfected COS-1 cells.

Amino Acid Sequence↗

PubMed1995

Identification and characterization of a functional promoter region in the human eosinophil IL-5 receptor alpha subunit gene.

The molecular basis for the commitment of multipotential myeloid progenitors to the eosinophil lineage, and the transcriptional mechanisms by which eosinophil-specific genes are subsequently expressed and regulated during eosinophil development are currently unknown. Interleukin-5 (IL-5) is a T cell and mast cell-derived cytokine with actions restricted to the eosinophil and closely related basophil lineages in humans. The high affinity receptor for IL-5 (IL-5R) is composed of an alpha subunit (IL-5R alpha) expressed by the eosinophil lineage, that associates with a beta c subunit shared with the receptors for IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF). As a prerequisite to studies of the transcriptional regulation of the IL-5R alpha subunit gene, we used three different methods, including primer extension, RNase protection, and 5'-RACE to precisely map the transcriptional start site to a position 15 base pairs (bp) upstream of the 5' end of the published sequence of IL-5R alpha exon 1. To initially identify the IL-5R alpha promoter, 3.5 kilobases (kb) and 561 bp of the 5' sequence flanking the transcriptional start site were subcloned into the promoterless pXP2-luciferase vector. Transient transfection of these constructs into an eosinophil-committed HL-60 subline, clone HL-60-C15, induced the expression of approximately 240-fold greater luciferase activity than the promoterless vector, identifying a strong functionally active promoter region within the 561 bp of sequence proximal to the transcriptional start site and with activity equivalent to pXP2 constructs containing the entire 3.5 kb of upstream sequence. To more precisely localize the cis-acting regulatory elements in this region important for promoter activity, a series of 5' deletion mutants of the 561-bp region were generated in the pXP2-luciferase vector. Deletion of the region between bp -432 and -398 reduced promoter activity by more than 80% in the HL-60-C15 cell line. Further analyses of the activity of the IL-5R alpha promoter constructs in various other eosinophil, myeloid, and non-myeloid cell lines indicated that the promoter was relatively myeloid and eosinophil lineage-specific in its expression. Consensus sequences for known transcription factor binding sites were not present in the 34-bp region of the promoter required for maximal activity, suggesting unique myeloid- and possibly eosinophil-specific regulatory elements.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1995

Characterization of critical residues in the cytoplasmic domain of the human interleukin-5 receptor alpha chain required for growth signal transduction.

Interleukin (IL)-5 binds to a cell surface receptor composed of two polypeptide chains, alpha and beta, both belonging to the hemopoietic cytokine receptor family. Mouse cells expressing common mouse beta chain (AIC2B) that were transfected with human IL-5 receptor (R)alpha cDNA proliferated in response to picomolar concentrations of human IL-5, indicating that a functional receptor was reconstituted. We show that in these cells, human (h)IL-5 as well as mouse (m)IL-3 induce tyrosine phosphorylation of beta chain and JAK 2 kinase. Phosphorylated beta receptor was co-precipitated with anti-JAK 2 antibodies, suggesting that both molecules were physically associated. IL-5 and IL-3 also induce cytosolic DNA binding activity as measured by an electrophoretic mobility shift assay using the interferon-gamma responsive region of human Fc gamma 1 gene DNA element. A deletion mutant of hIL-5R alpha lacking the cytoplasmic part could bind hIL-5 normally in association with the beta chain, but was unable to transmit a biological signal. The cytoplasmic domain was also indispensable for tyrosine phosphorylation and activation of DNA binding proteins. A membrane-proximal proline-rich element of the hIL-5R alpha cytoplasmic domain that is conserved among different members of the hemopoietic cytokine receptor family was essential for biological activity. Point mutations in this motif also knocked out IL-5-inducible JAK 2 phosphorylation.

Amino Acid Sequence↗

PubMed1995

Interleukin-5 and its receptor: a drug target for eosinophilia associated with chronic allergic disease.

A characteristic feature of chronic allergic diseases such as asthma is the increase in eosinophil numbers in the inflamed tissue. In light of its specificity for the development of eosinophils, interleukin-5 (IL-5) is considered the most important cytokine involved in the regulation of eosinophilia. Hence, an antagonist for IL-5 activity is a new target for drug discovery programs. We have examined the opportunity for both a random and a rational approach for the identification of such an antagonist. The elucidation of the structure of IL-5 and the initial structure/function analysis of the ligand/receptor complex constitute a first step towards the design of antagonistic compounds. The identification of a small compound by random screening able to inhibit the IL-5/IL-5 receptor interaction indicated an important domain in the receptor. We examine here protein-based IL-5 antagonists, such as IL-5-muteins, soluble IL-5 receptor constructs, and monoclonal antibodies, for their potential as IL-5/IL-5 receptor antagonists, and the use of a murine model of eosinophil airway inflammation for their evaluation.

Animals↗

PubMed1995

Structure/function analysis of human interleukin 5 and its receptor.

We have performed a detailed structure-function analysis of human interleukin 5 (hIL5) and its receptor. By testing a hIL5 mutant panel in a solid phase binding assay and a proliferation assay using hIL5 dependent cell-lines, areas on hIL5 involved in either the receptor alpha-subunit interaction or in receptor activation were identified. Epitope mapping data of a neutralizing and a non-neutralizing monoclonal antibody were in agreement with the mutant analysis. hIL5 binding areas on the IL5R alpha-subunit were identified by interspecies chimaera analysis. Finally, hIL5 mutants with reduced receptor activation potential have antagonistic properties.

Animals↗

PubMed1994

Three residues in the common beta chain of the human GM-CSF, IL-3 and IL-5 receptors are essential for GM-CSF and IL-5 but not IL-3 high affinity binding and interact with Glu21 of GM-CSF.

The beta subunit (beta c) of the receptors for human granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5) is essential for high affinity ligand-binding and signal transduction. An important feature of this subunit is its common nature, being able to interact with GM-CSF, IL-3 and IL-5. Analogous common subunits have also been identified in other receptor systems including gp130 and the IL-2 receptor gamma subunit. It is not clear how common receptor subunits bind multiple ligands. We have used site-directed mutagenesis and binding assays with radiolabelled GM-CSF, IL-3 and IL-5 to identify residues in the beta c subunit involved in affinity conversion for each ligand. Alanine substitutions in the region Tyr365-Ile368 in beta c showed that Tyr365, His367 and Ile368 were required for GM-CSF and IL-5 high affinity binding, whereas Glu366 was unimportant. In contrast, alanine substitutions of these residues only marginally reduced the conversion of IL-3 binding to high affinity by beta c. To identify likely contact points in GM-CSF involved in binding to the 365-368 beta c region we used the GM-CSF mutant eco E21R which is unable to interact with wild-type beta c whilst retaining full GM-CSF receptor alpha chain binding. Eco E21R exhibited greater binding affinity to receptor alpha beta complexes composed of mutant beta chains Y365A, H367A and I368A than to those composed of wild-type beta c or mutant E366A. These results (i) identify the residues Tyr365, His367 and Ile368 as critical for affinity conversion by beta c, (ii) show that high affinity binding of GM-CSF and IL-5 can be dissociated from IL-3 and (iii) suggest that Tyr365, His367 and Ile368 in beta c interact with Glu21 of GM-CSF.

Amino Acid Sequence↗

PubMed1994

Covalent modification of the interleukin-5 receptor by isothiazolones leads to inhibition of the binding of interleukin-5.

Using a fusion protein of the human interleukin-5-receptor alpha chain (hIL5R alpha) and the human IgG C gamma 3 chain (hIL5R alpha-h gamma 3), we have developed a solid-phase assay for high-flux screening of a collection of synthetic compounds. We report on the identification of isothiazolone derivatives as potent inhibitors of binding of interleukin-5 (IL5) to the hIL5R alpha, as measured in a solid-phase assay (soluble hIL5R alpha or hIL5R alpha-h gamma 3) or on COS-1 cells expressing the hIL5R alpha on the cell membrane. The binding of hIL4 and human granulocyte macrophage colony-stimulating factor (hGM-CSF) to their respective receptors is not inhibited by the isothiazolones in similar assay systems. Scatchard analysis revealed that these compounds caused a decrease in affinity of the IL5R alpha for IL5. The inhibition of binding IL5 to its receptor by the isothiazolone derivatives is abrogated by free-sulfhydryl-containing compounds such as dithiothreitol, indicating that the isothiazolones react with the sulfhydryl group of free cysteine residues in the hIL5R alpha. Mutation of Cys66 led to a receptor which still binds hIL5, but which was insensitive to the inhibition by isothiazolones. Mutation of Cys249 and Cys296 to serine resulted in complete loss of IL-5-binding activity. The use of radio-labeled isothiazolone confirmed that Cys66, present in the first domain of the receptor, is the target for covalent modification leading to a decrease in affinity.

Amino Acid Sequence↗

PubMed1994

Human tumor necrosis factor mutants with preferential binding to and activity on either the R55 or R75 receptor.

Previously, we reported that the cytotoxic activity of human (h) tumor necrosis factor (TNF) on murine (m) L929 cells requires the integrity of three loops (positions 30-36, 84-88 and 138-150) which cluster around the interface between each two subunits of the trimeric hTNF structure. The collection of hTNF mutants was further characterized by their activity on various human cell systems as well as by their binding to the two types of hTNF receptor (R), R55 and R75. It turned out that two amino acids (Leu29 and Arg32) were specifically involved in hR75 binding, as Leu29-->Ser (L29S) and Arg32-->Trp (R32W) mutant molecules had largely lost binding to hR75, but not to hR55. In order to screen for more highly R55-specific mutants, nine other amino acids were inserted at these two positions; only the substitutions L29G and L29Y showed an increased differential binding as compared to L29S, while no further improvement was found with mutations at position 32 compared to R32W. Biological assays mediated either by hR55 or hR75 confirmed the results obtained by physical binding to purified receptors. A similar substitution in mTNF, Arg32-->Tyr, also resulted in a preferential loss of binding to hR75 and a large decrease in mR75-mediated bioactivity. Except for the double mutant L29S-R32W, all other tested amino acid substitutions in the loops at positions 30-36 or 84-88 of hTNF led to a substantial loss of affinity for both receptors and a concomitant reduction of biological activity. In the loop at positions 138-150, the non-conservative replacement of Glu by Lys at position 146 (E146K) resulted in an even lower binding to R75 as compared to R32W, while binding on and bioactivity through R55 was only slightly reduced. Remarkably, a reversed differential binding was observed after substitution at position 143 in hTNF; replacing Asp by non-conservative residues such as Tyr, Phe or Asn resulted in a much larger decrease in binding to R55 than to R75. In conclusion, receptor-specific mutants such as R32W, E146K and D143N can be used to study the function either of R55 or R75 on different human cell types. In vivo, we presume that the R55-specific mutants will retain antitumor activity in the absence of R75-dependent, severe side effects.

Animals↗

PubMed1994

Dissociation of TNF-alpha cytotoxic and proinflammatory activities by p55 receptor- and p75 receptor-selective TNF-alpha mutants.

Human tumour necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine capable of killing mammalian tumour cells in vitro and in vivo, and of enhancing the proinflammatory activity of leucocytes and endothelium, the latter effects limiting its usage as an antitumour agent in humans. Using TNF-alpha mutants with a selective capacity to bind to the TNF p55 receptor (TNFR55) or to the p75 receptor (TNFR75) we show here that these two major activities of TNF-alpha can be dissociated. The TNFR55-selective mutants (R32W, E146K and R32W-S86T) which bind poorly to TNFR75 displayed similar potency to wild-type TNF in causing cytotoxicity of a human laryngeal carcinoma-derived cell line (HEp-2) and cytostasis in a human leukaemic cell line (U937). However, these TNFR55-selective mutants exhibited lower proinflammatory activity than wild-type TNF. Specifically, TNF-alpha's priming of human neutrophils for superoxide production and antibody-dependent cell-mediated cytotoxicity, platelet-activating factor synthesis and adhesion to endothelium were reduced by up to 170-fold. Activation of human endothelial cell functions represented by human umbilical venular endothelial cell (HUVEC) adhesiveness for neutrophils, E-selectin expression, neutrophil transmigration and IL-8 secretion were also reduced by up to 280-fold. On the other hand, D143F, a TNFR75-selective mutant tested either alone or in combination with TNFR55-selective mutants, did not stimulate these activities despite being able to cause cytokine production in TNFR75-transfected PC60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1994

Interleukin 5 synthesis by eosinophils: association with granules and immunoglobulin-dependent secretion.

Interleukin 5 (IL-5) is the main factor that promotes the terminal differentiation of eosinophil progenitors (as indicated by colony formation assays), and enhances the effector capacity of mature eosinophils. IL-5 is produced by T lymphocytes, CD4-/CD8- and mast cells and recently, messenger (m)RNA of this cytokine has been identified in eosinophils from patients with coeliac disease, asthma, or eosinophilic heart diseases. In this study, IL-5 mRNA and immunoreactive IL-5 protein were detected in tissue and blood eosinophils from patients with eosinophilic cystitis or hypereosinophilic syndromes but not in Crohn's disease. By electron microscopy associated to immunogold staining, immunoreactive IL-5 was identified in eosinophilic granules. After stimulation with IgA-, IgE-, or IgG-immune complexes, blood eosinophils were shown, by immunocytochemistry and by enzyme-linked immunosorbent assay, to secrete IL-5. These observations demonstrate that eosinophils, under physiological stimulation, can release significant amounts of IL-5, which may contribute to local eosinophil recruitment and activation.

Cytoplasmic Granules↗

PubMed1994

Structure-activity studies of human tumour necrosis factors.

The mechanism by which tumour necrosis factors (TNF and lymphotoxin, also called TNF alpha and TNF beta respectively) exert their cytotoxic activity on many malignant cells, remains largely unknown. Furthermore, the broad array of differentiation (gene induction) and mitogenic activities towards many primary cells is still a subject of intensive investigation. TNF is an important mediator in inflammation, immune responses and infection-related phenomena and these activities contribute to the severe toxicity seen when TNF is used as an anticancer agent. The first step in the mechanism of action is the specific binding of the ligand to its receptors and dissection of the molecular mechanism involved in this interaction is the subject of this review. The reasons for the interest in this aspect are obvious: first, the development of strong antagonistic TNF analogues can be useful in dampening the potentially lethal or debilitating effects of an overproduction of the cytokine (as in septic shock or rheumatoid arthritis). Secondly, since two distinct TNF receptors exist, construction of TNF muteins that distinguish between both types may lead to derivatives of this pleiotropic agent with a more restricted biological activity pattern. Ideally, one would like to develop a TNF mutant that has retained its cytotoxic action on tumour cells without inducing the deleterious systemic toxicity. Such an optimized TNF molecule could become a potent anticancer agent.

Amino Acid Sequence↗