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Biomedical subjects

M A Vadas

Publications and source records attributed to M A Vadas.

At least 19 recordsLinked to original sources
PubMed1997

Roles of the N and C terminal domains of the interleukin-3 receptor alpha chain in receptor function.

The interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and IL-5 receptor alpha chains are each composed of three extracellular domains, a transmembrane domain and a short intracellular region. Domains 2 and 3 constitute the cytokine receptor module (CRM), typical of the cytokine receptor superfamily; however, the function of the N-terminal domain is not known. We have investigated the functions of the N-terminal and C-terminal domains of the IL-3 receptor (IL-3R) alpha chain. We find that cells transfected with the receptor beta chain (h beta c) and a truncated IL-3R alpha that is devoid of the intracellular region fail to proliferate or to activate STAT5 in response to human IL-3, despite binding the IL-3 with affinity indistinguishable from that of full-length receptor. In addition, IL-3-induced phosphorylation of h beta c was not detected. Thus, the IL-3R alpha intracellular region does not contribute detectably to stabilization of the receptor/ligand complex, but is essential for signal propagation. In contrast, a truncated IL-3R alpha with the N-terminal domain deleted interacts functionally with the beta chain; mouse cells transfected with these receptor chains proliferate in response to human IL-3 and STAT5 transcription factor is activated. High- and low-affinity binding sites are retained, although the affinity for IL-3 is decreased 15-fold, indicating a significant role for the N-terminal domain in IL-3 binding.

Amino Acid Sequence↗
PubMed1997

Lumen formation during angiogenesis in vitro involves phagocytic activity, formation and secretion of vacuoles, cell death, and capillary tube remodelling by different populations of endothelial cells.

BACKGROUND: We have utilised an in vitro model of angiogenesis to investigate the morphological changes which occur during the formation of a lumen in capillary tubes. METHODS AND RESULTS: On collagen 1 gel in the presence of phorbol myristate acetate (PMA) and anti-alpha 2 beta 1 antibody, cell aggregation and alignment takes place within two hours of plating. The initial apparently homogeneous population of endothelial cells (EC) actually display at least three distinct phenotypes. One population, characterised by a phagocytic phenotype, migrated through the gel creating channels and defines the extent of the capillary network. These are later enveloped by a second population of cells characterised by intracellular vacuoles. The ultimate fate of these vacuoles is fusion with the plasma membrane. By 12 hours the original phagocytic cell population undergoes cell death, which morphologically appears apoptotic in nature. A consequence of the secretion of vacuoles and programmed cell death is the extensive remodelling of the capillary tubes, resulting in expansion of the intercellular space into a lumen. The remodelling results in 45% of the EC membrane contacting the lumenal surface at the expense of EC-EC and EC-matrix contact. A third population of cells implant between the EC involved in lumen formation and thus expand the size of the capillary tube. CONCLUSION: Thus, in the formation of a mature multicellular lumen we have identified a number of key events. First, cell-cell contact is essential in order to define the intercellular space. Second, at least three morphologically distinct subpopulations of ECs are involved. Third, vacuole formation and programmed cell death are required for expansion of the intercellular space which ultimately becomes the lumen.

Antibodies↗
PubMed1997

A T cell-specific enhancer in the interleukin-3 locus is activated cooperatively by Oct and NFAT elements within a DNase I-hypersensitive site.

Interleukin-3 (IL-3) is a cytokine that is expressed primarily in activated T cells. Here we identified an inducible T cell-specific enhancer 14 kb upstream of the IL-3 gene that responded to activation of T cell receptor signaling pathways. The IL-3 enhancer spanned an inducible cyclosporin A-sensitive DNase I-hypersensitive site found only in T cells. Four NFAT-like elements exist within the enhancer. The two most active NFAT-like elements were located at the center of the DNase I-hypersensitive site. One of these NFAT-like elements encompassed overlapping Oct- and NFATp/c-binding sites, which functioned in a highly synergistic manner. We suggest that the T cell-specific expression of the IL-3 gene is partly controlled through the enhancer by cooperation between Oct and NFAT family proteins.

Base Sequence↗
PubMed1997

Regulation of leucocyte-endothelial interactions of special relevance to atherogenesis.

1. The cellular events underlying atherosclerosis include the accumulation of lipid-laden monocytes in the neointima. This process is associated with the expression of adhesion proteins and chemokines by the endothelium, in a manner similar to that seen after the administration of pro-inflammatory cytokines to endothelial cells. 2. The processes that limit endothelial responses to proinflammatory cytokines are, therefore, the subject of this paper. Evidence is presented that the cytokine TGF-beta exerts a tonic inhibitory influence on endothelial responses. Furthermore, the smooth muscle cells adjacent to endothelial cells have a similar effect to exogenous TGF-beta and this suggests that these two cells form a functional interactive unit. Finally, the atheroprotective lipid fraction, high-density lipoproteins (HDL), also inhibits endothelial activation. The mechanism of effect of HDL that appears separate from its traditional role in cholesterol transport may yield novel insights into atheroprotection.

Animals↗
PubMed1997

Signals for activation of the GM-CSF promoter and enhancer in T cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the many cytokines produced following T-cell activation. It is also produced in a variety of other cell types, in particular following activation by inflammatory mediators. Changes in the rate of transcription are important in the control of GM-CSF expression in T cells and in fibroblasts and endothelial cells. The GM-CSF gene contains two distinct transcriptional control regions. These are the proximal promoter consisting of the first 120 bp from the transcription start site and an enhancer located approximately 3 kb upstream from the proximal promoter. Distinct regions of the proximal promoter respond to a wide array of signals such as phorbol myristate acetate (PMA) and Ca2+ ionophore or phytohemaglutinin (PHA), CD28 activation, human T leukemia virus (HTLV)-1 tax, TNF, and interleukin 1 (IL-1). The transcription factors that mediate these responses have mainly been defined, with the major inducible proteins being the NF-kappa B/rel and AP-I families of transcription factors. In contrast to the promoter, the enhancer responds only to PMA and Ca2+ ionophore signals and binds NFAT/AP-1 complexes that appear to mediate its function.

Animals↗
PubMed1996

A discontinuous eight-amino acid epitope in human interleukin-3 binds the alpha-chain of its receptor.

We have previously reported that, within the first helix of human interleukin (IL)-3, residues Asp21 and Glu22 are important for interaction with the alpha- and beta-chains of the IL-3 receptor, respectively. In order to define more precisely the sites of interaction with the receptor, we have performed molecular modeling of the helical core of IL-3 and single amino acid substitution mutagenesis of residues predicted to lie on the surfaces of the A, C, and D helices. The resulting analogues were characterized for their abilities to stimulate proliferation of TF-l cells and for binding to the high affinity (alpha- and beta-chain; IL-3Ralpha/Rbeta) or low affinity (alpha-chain alone; IL-3Ralpha) IL-3 receptor. We found that in addition to Asp21, residues Ser17, Asn18, and Thr25 within the A helix and Arg108, Phe113, Lys116, and Glu119 within the D helix of IL-3 were important for biological activity. Analysis of their binding characteristics revealed that these analogues were deficient in binding to both the IL-3Ralpha/Rbeta and the IL-3Ralpha forms of the receptor, consistent with a selective impairment of interaction with IL-3Ralpha. Molecular modeling suggests that these eight amino acid residues are adjacent in the tertiary structure, consistent with a discontinuous epitope interacting selectively with IL-3Ralpha. On the other hand, Glu22 of IL-3 was found to interact preferentially with the beta-chain with bulky and positively charged substitutions causing greater than 10,000-fold reduction in biological activity. These results show fundamental differences between IL-3 and granulocyte-macrophage colony-stimulating factor in the structural basis for recognition of their receptors that has implications for the construction of novel analogues and our understanding of receptor activation.

Amino Acid Sequence↗
PubMed1996

Differential regulation of the stability of cytokine mRNAs in lipopolysaccharide-activated blood monocytes in response to interleukin-10.

Adenosine-uridine (AU) instability elements, found in the 3'-untranslated regions of numerous mRNAs, target these mRNAs for rapid degradation. In addition, the degradation rate of some mRNAs that contain AU instability elements can change. This modulation of mRNA stability is an important component in the regulation of expression of many of the cytokines that control the production and function of blood cells. However, it has not been clear whether the stabilities of individual cytokine mRNAs that contain AU instability elements are coordinately regulated or whether different mRNAs can be independently regulated. We have investigated the influence of the cytokine synthesis inhibitory factor interleukin (IL)-10 on the turnover of granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), and IL-10 mRNAs in human blood monocytes stimulated with lipopolysaccharide. We find that all three mRNAs are destabilized in response to IL-10 but at different times. The G-CSF and GM-CSF mRNAs respond similarly, being rapidly destabilized, consistent with a direct influence of IL-10 receptor-mediated signals on the stability of these mRNAs. In contrast the IL-10 mRNA became unstable only after several hours of treatment with IL-10, suggesting that the IL-10 mRNA, although it also contains AU instability elements, is not co-regulated with the G-CSF and GM-CSF mRNAs but is regulated by a secondary factor produced in response to IL-10.

Gene Expression Regulation↗
PubMed1996

Transforming growth factor-beta 1 inhibits the production of IL-8 and the transmigration of neutrophils through activated endothelium.

A central mechanism of inflammation is the activation of vascular endothelium by the inflammatory cytokines TNF-alpha and IL-1. These cytokines induce the expression of adhesion molecules, the elaboration of chemokines, and the transendothelial migration of white cells. TGF-beta 1 has anti-inflammatory properties, is expressed in the vessel wall, and has previously been shown to inhibit leukocyte adhesiveness to the endothelium at least in part by inhibiting the expression of E-selectin. We now show that TGF-beta 1 also inhibits the migration of neutrophils through endothelial monolayers activated by TNF-alpha. At a dose of 10 U/ml TNF-alpha, the transmigration of neutrophils was inhibited 42.7 +/- 7.9% (n = 8) by 0.2 ng/ml TGF-beta 1. Furthermore, TGF-beta 1 inhibited, in a time- and dose-dependent fashion, the elaboration of IL-8 by TNF-activated endothelial cells by between 33 and 78% (TNF doses from 100 down to 0.1 U/ml) and the elaboration of mRNA for IL-8 by 69%. TGF-beta 1 treatment did not significantly alter the TNF-induced IL-8 mRNA stability, suggesting that the mechanism of action of TGF-beta 1 is on gene transcription. Neutrophil transmigration through cytokine-activated endothelium involves both IL-8-dependent and IL-8-independent mechanisms. Using an anti-IL-8 Ab, we show that TGF-beta 1 inhibits only the IL-8-dependent pathway, but does not affect the IL-8-independent transendothelial migration mechanism. These and our previous results show that TGF-beta1, achieves its anti-inflammatory properties by inhibiting the expression of at least two genes, E-selectin and IL-8, which are essential in the inflammatory pathway.

Cell Movement↗
PubMed1996

Cold shock domain proteins repress transcription from the GM-CSF promoter.

The human granulocyte-macrophage colony stimulating factor (GM-CSF) gene promoter binds a sequence-specific single-strand DNA binding protein termed NF-GMb. We previously demonstrated that the NF-GMb binding sites were required for repression of tumor necrosis factor-alpha (TNF-alpha) induction of the proximal GM-CSF promoter sequences in fibroblasts. We now describe the isolation of two different cDNA clones that encode cold shock domain (CSD) proteins with NF-GMb binding characteristics. One is identical to the previously reported CSD protein dbpB and the other is a previously unreported variant of the dbpA CSD factor. This is the first report of CSD factors binding to a cytokine gene. Nuclear NF-GMb and expressed CSD proteins have the same binding specificity for the GM-CSF promoter and other CSD binding sites. We present evidence that CSD factors are components of the nuclear NF-GMb complex. We also demonstrate that overexpression of the CSD proteins leads to complete repression of the proximal GM-CSF promoter containing the NF-GMb/CSD binding sites. Surprisingly, we show that CSD overexpression can also directly repress a region of the promoter which apparently lacks NF-GMb/CSD binding sites. NF-GMb/CSD factors may hence be acting by two different mechanisms. We discuss the potential importance of CSD factors in maintaining strict regulation of the GM-CSF gene.

Amino Acid Sequence↗
PubMed1996

Extracellular truncations of h beta c, the common signaling subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5, lead to ligand-independent activation.

The hypothesis that extracellular truncation of the common receptor subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and IL-5 (h beta c) can lead to ligand-independent activation was tested by infecting factor-dependent hematopoietic cell lines with retroviruses encoding truncated forms of h beta c. A truncation, resembling that in v-Mpl, and retaining 45 h beta c-derived extracellular residues, led to constitutive activation in the murine myeloid cell line, FDC-P1. However, infection of cells with retrovirus encoding a more severely truncated receptor, retaining only 7 h beta c-derived extracellular residues, did not confer factor independence on these cells. These experiments show that truncation activates the receptor and define a 37-amino acid segment of h beta c (H395-A431) which contains two motifs conserved throughout the cytokine receptor superfamily (consensus Y/H XX R/Q VR and WSXWS), as essential for factor-independent signaling. The mechanism of activation was also investigated in less severe truncations. A receptor that retains the entire membrane-proximal domain (domain 4) also conferred factor independent growth on FDC-P1 cells; however, a retrovirus encoding a truncated form of h beta c having two intact membrane proximal domains did not have this ability, suggesting that domain 3 may have an inhibitory role in h beta c. The ability of these receptors to confer factor independence was cell specific as demonstrated by their inability to confer factor-independent growth when introduced into the murine IL-3-dependent pro-B cell line BaF-B03. These results are consistent with a model in which activation requires unmasking of an interactive receptor surface in domain 4 and association with a myeloid-specific receptor or accessory component. We suggest that in the absence of ligand intramolecular interactions prevent inappropriate signaling.

Animals↗
PubMed1996

Chronic expression of P-selectin on endothelial cells stimulated by the T-cell cytokine, interleukin-3.

P-selectin expressed on the surface of endothelium mediates leukocyte adhesion in vitro and rolling in vivo. Several inducers of cell-surface P-selectin expression on endothelial cells (EC) have previously been identified, all of which yield transient cell-surface expression of P-selectin lasting minutes to a few hours. We now show that a T-lymphocyte product, interleukin-3 (IL-3), stimulates the long-term endothelial cells (HUVEC). IL-3 induced cell-surface P-selectin expression in two phases. An initial peak at 10 minutes was followed by a prolonged upregulation beginning 16 hours after IL-3 addition and lasting at least 4 days. The level of P-selectin expression induced by IL-3 added for 48 hours was similar to that induced by treatment of HUVEC for 10 minutes with thrombin, and the effect of adding IL-3 for 48 hours followed by thrombin for 10 minutes was additive. Induction of cell-surface P-selectin expression by IL-3 was blocked by pretreatment of EC with a blocking monoclonal antibody against the IL-3 receptor alpha-chain. Lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) and a mutant form of IL-3 with decreased potency did not induce cell-surface P-selectin expression after 48 hours' incubation with HUVEC, suggesting that the effect was specific to IL-3. The increase in cell-surface P-selectin expression occurring after 16 hours of incubation with IL-3 was accompanied by a similar prolonged increase in the steady-state mRNA level that was not observed at 10 minutes after IL-3 addition. As T-lymphocyte infiltration is a hallmark of chronic inflammation, our observations suggest that the secretion of IL-3 by T lymphocytes may serve to maintain the inflammatory state during chronic inflammation.

Cells, Cultured↗
PubMed1996

Monoclonal antibody 7G3 recognizes the N-terminal domain of the human interleukin-3 (IL-3) receptor alpha-chain and functions as a specific IL-3 receptor antagonist.

The human interleukin-3 receptor (IL-3R) is expressed on myeloid, lymphoid, and vascular endothelial cells, where it transduces IL-3-dependent signals leading to cell activation. Although IL-3R activation may play a role in hematopoiesis and immunity, its aberrant expression or excessive stimulation may contribute to pathologic conditions such as leukemia, lymphoma, and allergic reactions. We describe here the generation and characterization of a monoclonal antibody (MoAb), 7G3, which specifically binds to the IL-3R alpha-chain and completely abolishes its function. MoAb 7G3 immunoprecipitated and recognized in Western blots the IL-3R alpha-chain expressed by transfected cells and bound to primary cells expressing IL-3R alpha. MoAb 7G3 bound the IL-3R alpha-chain with a kd of 900 pmol/L and inhibited 125I-IL-3 binding to high- and low-affinity receptors in a dose-dependent manner. Conversely, IL-3 but not granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited 125I-7G3 binding to high- and low-affinity IL-3Rs, indicating that MoAb 7G3 and IL-3 bind to common or adjacent sites. In keeping with the inhibition of IL-3 binding, MoAb 7G3 antagonized IL-3 biologic activities, namely stimulation of TF-1 cell proliferation, basophil histamine release, and IL-6 and IL-8 secretion from human endothelial cells. Two other anti-IL-3R alpha-chain MoAbs failed to inhibit IL-3 binding or function. Epitope mapping experiments using truncated IL-3R alpha-chain mutants and IL-3R alpha/GM-CSFR alpha chimeras revealed that 31 amino acids in the N-terminus of IL-3R alpha were required for MoAb 7G3 binding. MoAb 7G3 may be of clinical significance for antagonizing IL-3 in pathologic conditions such as some myeloid leukemias, follicular B-cell lymphoma, and allergy. Furthermore, these results implicate the N-terminal domain of IL-3R alpha in IL-3 binding. Since this domain is unique to the IL-3/GM-CSF/IL-5 receptor subfamily, it may represent a novel and common binding feature in these receptors.

Animals↗
PubMed1996

IL-3 receptor expression, regulation and function in cells of the vasculature.

IL-3 is a haemopoietic growth factor which stimulates the production and functional activity of various blood cell types. Recent evidence suggests that the target cell population of IL-3 is not restricted to haemopoietic cells as previously thought, but vascular cells such as endothelial cells also express receptors for and respond to this cytokine. Interestingly, IL-3 was found to regulate endothelial responses related to inflammation, immunity and haemopoiesis. These findings, summarized in this review, offer new insight into the physiological function of IL-3 and may also be of clinical importance, as IL-3 is used in bone marrow reconstitution following cancer therapy.

Cells, Cultured↗
PubMed1995

Neutrophils activated by granulocyte-macrophage colony-stimulating factor express receptors for interleukin-3 which mediate class II expression.

Freshly isolated peripheral blood neutrophils, unlike monocytes and eosinophils, do not bind interleukin-3 (IL-3) or respond to IL-3). We show that neutrophils cultured for 24 hours in granulocyte-macrophage colony-stimulating factor (GM-CSF) express mRNA for the IL-3 receptor (R) alpha subunit, as shown by RNase protection assays, and IL-3R alpha chain protein, as shown by cytometric analysis using two different specific monoclonal antibodies. This effect was selective for GM-CSF, because granulocyte colony-stimulating factor, tumor necrosis factor-alpha, interferon-gamma, and IL-1 failed to induce the IL-3 receptor. Saturation binding curves with 125I-IL-3 and Scatchard transformation showed the presence of about 100 high-affinity and 4,000 low-affinity receptors. Because neutrophils have been shown to express human leukocyte antigen (HLA)-DR in response to GM-CSF, we examined the possibility that IL-3 could augment HLA-DR expression on GM-CSF-treated cells. We found that neutrophils incubated with 30 ng/mL IL-3 as well as 0.1 ng/mL GM-CSF expressed a mean of 2.1-fold higher levels of HLA-DR than with GM-CSF alone (P < .005), confirming the signaling competence of the newly expressed IL-3R. This increase was seen even at maximal concentrations of GM-CSF and IL-3 can have an additive effect on mature human cells. The augmentation of HLA-DR by IL-3 was specific because it could be inhibited by a blocking anti-IL-3R antibody. Expression of class II molecules by neutrophils under these conditions may have significance for antigen presentation. These results provide further evidence for the role of GM-CSF as an amplification factor in inflammation by inducing neutrophil responsiveness to IL-3 produced by T cells or mast cells.

Cells, Cultured↗
PubMed1995

Regulation of granulocyte-macrophage colony-stimulating factor and E-selectin expression in endothelial cells by cyclosporin A and the T-cell transcription factor NFAT.

Nuclear factor of activated T cells (NFAT) was originally described as a T-cell-specific transcription factor athat supported the activation of cytokine gene expression and mediated the immunoregulatory effects of cyclosporin A (CsA). As we observed that activated endothelial cells also expressed NFAT, we tested the antiinflammatory properties of CsA in endothelial cells. Significantly, CsA completely suppressed the induction of NFAT in endothelial cells and inhibited the activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) gene regulatory elements that use NFAT by 60%. CsA similarly mediated a reduction of up to 65% in GM-CSF mRNA and protein expression in activated endothelial cells. CsA also suppressed E-selectin, but not vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells, even though the E-selectin promoter is activated by NF-kappa B rather than NFAT. Hence, induction of cell surface expression of this leukocyte adhesion molecule by tumor necrosis factor (TNF)-alpha was reduced by 40% in the presence of CsA, and this was reflected by a 29% decrease in neutrophil adhesion. The effects of CsA on endothelial cells were also detected at the chromatin structure level, as DNasel hypersensitive sites within both the GM-CSF enhancer and the E-selectin promoter were suppressed by CsA. This represents the first report of NFAT in endothelial cells and suggests mechanisms by which CsA could function as an antiinflammatory agent.

Animals↗
PubMed1995

Interferon-gamma upregulates interleukin-3 (IL-3) receptor expression in human endothelial cells and synergizes with IL-3 in stimulating major histocompatibility complex class II expression and cytokine production.

The human interleukin-3 (IL-3) receptor is constitutively expressed on certain hematopoietic cells where it mediates proliferation and differentiation, or functional activation. We have recently found that human umbilical vein endothelial cells (HUVECs) also express IL-3 receptors and that the expression is enhanced by stimulation with the monokine tumor necrosis factor alpha. In this report we show that the lymphokine interferon gamma (IFN gamma) is a powerful stimulator of the IL-3 receptor of HUVECs and that the combination of IL-3 and IFN gamma has a synergistic effect on major histocompatibility complex (MHC) class II expression and on the production of the early-acting hematopoietic cytokines IL-6 and granulocyte colony-stimulating factor (G-CSF). IFN gamma caused a time- and dose-dependent up-regulation of mRNA for both the alpha and beta chains of the IL-3 receptor, with maximal effects occurring 12 to 24 hours after stimulation with IFN gamma at 100 U/mL. Induction of mRNA correlated with protein expression on the cell surface, as judged by monoclonal antibody staining of both receptor chains and by the ability of HUVEC to specifically bind 125I-labeled IL-3 (125I-IL-3). Scatchard analysis of HUVECs stimulated with IFN gamma at 100 U/mL for 24 hours showed approximately 6,300 IL-3 receptors per cell that were of a high affinity class (dissociation constant [kd] = 500 pmol/L) only. The addition of IL-3 to IFN gamma-treated HUVECs strongly enhanced the expression of MHC class II antigen. Importantly, IFN gamma and IL-3 also exhibited a synergistic effect in the induction of the mRNA for G-CSF and IL-6. This was reflected in increased amounts of G-CSF and IL-6 protein in HUVEC supernatants. In contrast, IFN gamma and IL-3 did not stimulate granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-8 production in HUVECs. These results show that IFN gamma is a strong stimulator of IL-3 receptor expression in HUVECs and suggest that in vivo T-cell activation, causing the concomitant production of IFN gamma and IL-3, may lead to enhanced endothelial MHC class II expression and to the selective production of early-acting hematopoietic cytokines. Thus, IL-3 could influence immunity and hematopoiesis by acting not only on hematopoietic cells, but also on vascular endothelium.

Drug Synergism↗