Allergenic proteases cleave the chemokine CX3CL1 directly from the surface of airway epithelium and augment the effect of rhinovirus.

CX3CL1 has been implicated in allergen-induced airway CD4+ T-lymphocyte recruitment in asthma. As epidemiological evidence supports a viral infection-allergen synergy in asthma exacerbations, we postulated that rhinovirus (RV) infection in the presence of allergen augments epithelial CX3CL1 release. Fully differentiated primary bronchial epithelial cultures were pretreated apically with house dust mite (HDM) extract and infected with rhinovirus-16 (RV16). CX3CL1 was measured by enzyme-linked immunosorbent assay and western blotting, and shedding mechanisms assessed using inhibitors, protease-activated receptor-2 (PAR-2) agonist, and recombinant CX3CL1-expressing HEK293T cells. Basolateral CX3CL1 release was unaffected by HDM but stimulated by RV16; inhibition by fluticasone or GM6001 implicated nuclear factor-κB and ADAM (A Disintegrin and Metalloproteinase) sheddases. Conversely, apical CX3CL1 shedding was stimulated by HDM and augmented by RV16. Although fluticasone or GM6001 reduced RV16+HDM-induced apical CX3CL1 release, heat inactivation or cysteine protease inhibition completely blocked CX3CL1 shedding. The HDM effect was via enzymatic cleavage of CX3CL1, not PAR-2 activation, yielding a product mitogenic for smooth muscle cells. Extracts of Alternaria fungus caused similar CX3CL1 shedding. We have identified a novel mechanism whereby allergenic proteases cleave CX3CL1 from the apical epithelial surface to yield a biologically active product. RV16 infection augmented HDM-induced CX3CL1 shedding-this may contribute to synergy between allergen exposure and RV infection in triggering asthma exacerbations and airway remodeling.

Systematic chemical and molecular profiling of MLL-rearranged infant acute lymphoblastic leukemia reveals efficacy of romidepsin.

To address the poor prognosis of mixed lineage leukemia (MLL)-rearranged infant acute lymphoblastic leukemia (iALL), we generated a panel of cell lines from primary patient samples and investigated cytotoxic responses to contemporary and novel Food and Drug Administration-approved chemotherapeutics. To characterize representation of primary disease within cell lines, molecular features were compared using RNA-sequencing and cytogenetics. High-throughput screening revealed variable efficacy of currently used drugs, however identified consistent efficacy of three novel drug classes: proteasome inhibitors, histone deacetylase inhibitors and cyclin-dependent kinase inhibitors. Gene expression of drug targets was highly reproducible comparing iALL cell lines to matched primary specimens. Histone deacetylase inhibitors, including romidepsin (ROM), enhanced the activity of a key component of iALL therapy, cytarabine (ARAC) in vitro and combined administration of ROM and ARAC to xenografted mice further reduced leukemia burden. Molecular studies showed that ROM reduces expression of cytidine deaminase, an enzyme involved in ARAC deactivation, and enhances the DNA damage-response to ARAC. In conclusion, we present a valuable resource for drug discovery, including the first systematic analysis of transcriptome reproducibility in vitro, and have identified ROM as a promising therapeutic for MLL-rearranged iALL.

The generation and characterisation of antagonist RNA aptamers to MCP-1.

Monocyte chemoattractant protein-1 (MCP-1) has been implicated as a powerful pro-inflammatory mediator and may represent a potentially important, therapeutic opportunity for treatment of inflammatory disease and atherosclerosis. To further investigate the role of MCP-1 in inflammatory disorders we have isolated a series of RNA aptamers that bind specifically to mouse MCP-1. The highest affinity aptamers, designated ADR7 and ADR22, have been functionally characterised in vitro and in cell based assays. ADR7 and ADR22 have an affinity of 180 pM and 370 pM respectively for mouse MCP-1, they can antagonise MCP-1 binding to heparin and specifically antagonise MCP-1 induced chemotaxis in a cell based assay. An interesting feature of ADR22 but not ADR7 is that it is capable of antagonising the function of human MCP-1, demonstrating the high level of specificity of these aptamers and that the aptamers recognise MCP-1 in different ways. The aptamers may be used as a tool to further investigate the role of MCP-1 in inflammatory disorders and may also have a role as a therapeutic agent.

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands.

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1-4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono-fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.

A time-resolved immunofluorometric method for the measurement of sialyl Lewis x-synthesizing alpha1,3-fucosyltransferase activity.

We describe here an assay that employs a highly sensitive nonradioactive method, time-resolved fluorometry, for measuring the activity of the enzyme GDP-Fuc:NeuNAcalpha2-3Galbeta1-4GlcNAc-R (Fuc to GlcNAc) alpha1,3-fucosyltransferase (alpha1,3FT). In this assay, a neoglycoprotein substrate of alpha1,3FT is immobilized on a microtiter plate. Incubation with the fucose donor GDP-fucose and enzyme source converts the acceptor NeuNAcalpha2-3Galbeta1-4GlcNAc-R to the product NeuNAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc-R, which is quantified using a product-specific (antisialyl Lewis x) primary antibody and europium chelate-labeled secondary antibody. In the development of the assay, we used extracts of alpha1,3FT-transfected insect cells as the specific enzyme source. The reaction product formation was proportional to time of incubation (0-2 h) and the extract added (0.1-10 microU of enzyme) and was dependent on the GDP-fucose and glycoconjugate acceptor. We have also demonstrated with different cultured cancer cell lines that this time-resolved immunofluorometric assay allows rapid measurement of alpha1,3FT activity from a large number of crude cell lysate samples. Our results indicated that cell lines which expressed more sialyl Lewis x determinant on their surfaces had higher levels of alpha1,3FT activity. The advantages of this new assay are high sensitivity and a wide linear range of measurement. The assay is expected to be useful in the determination of regulation mechanisms of sialyl Lewis x-synthesizing alpha1,3-fucosyltransferases.

Receptor mediated endocytosis and intracellular fate of interleukin 1.

We have studied the receptor mediated endocytosis of interleukin 1 (IL1) by the murine thymoma cell line EL4. These cells express the Type I IL1 receptor which binds its ligand with both high (Kd = 65 pM) and low affinity (Kd = 14.5 nM). We have shown that the two affinity states of the receptor have different rates of turnover both in the absence and presence of ligand. The biological responses of cells to IL1 stimulation are rapid and occur at low levels of receptor occupancy, whereas receptor mediated endocytosis of IL1 is relatively slow. Internalized IL1 appears to accumulate within cells in a non-degraded form and a proportion of this is associated with a detergent insoluble intracellular fraction, which may reflect transport to the nucleus. In this article, we review our previous findings and discuss the possible biological significance of IL1 internalization and nuclear targeting.

Acute upregulation of interleukin-1 receptor by ligand.

In this study we have investigated the effect that interleukin 1 (IL-1) has on cell surface IL-1 receptor expression in the murine thymoma cell line, EL4 6.1. These cells express IL-1 receptors with both high affinity (Kd = 65 pM, 986 receptors/cell) and low affinity (Kd = 14.5 nM, 10,417 receptors/cell). The high- and low-affinity receptors are indistinguishable by crosslinking studies performed at both high and low ligand concentrations. However, the two affinity states could be functionally distinguished on the basis of their internalization of ligand. Receptor-mediated endocytosis was dependent upon the concentration of ligand bound to the cells. In the presence of low IL-1 concentrations receptor-mediated endocytosis was slow, whereas at high IL-1 concentrations, endocytosis was more rapid. Furthermore, receptor-mediated endocytosis of IL-1 did not result in downregulation of surface IL-1 receptors. Indeed, both kinetic and equilibrium binding studies revealed that pre-incubation of cells with IL-1 alpha resulted in an acute upregulation of 125IL-1 alpha binding to high affinity surface receptors in a time and energy dependent manner. Examination of the association kinetics suggested that increased binding was not attributable to positive co-operativity of the high affinity IL-1 receptor, but was due to increasing IL-1 receptor number. This observation was confirmed by equilibrium binding studies. Moreover, receptor numbers were not enhanced by de novo synthesis, nor release of receptors from an intracellular pool. The observed increases in surface ligand binding were most probably due to conversion of the surface pool of low affinity receptors into high affinity receptors.

Investigation of guanine-nucleotide-binding protein involvement and regulation of cyclic AMP metabolism in interleukin 1 signal transduction.

The involvement of guanine-nucleotide-binding proteins (G-proteins) and regulation of cyclic AMP (cAMP) in interleukin 1 (IL1) signal transduction has been investigated in EL4 and 7OZ/3 cells expressing Type 1 and Type 2 IL1 receptors respectively. Results show that in both cell types IL1 alone failed to induce changes in cellular cAMP levels, and in membrane preparations the cytokine had no significant effect on adenylate cyclase activity. In contrast, forskolin stimulated cAMP levels in cells and membranes. IL1 did not significantly alter GTPase activity or rate of guanosine 5'-[gamma-[35S]thio]triphosphate binding measured in membrane preparations from the EL4 and 7OZ/3 cells. In EL4-cell membrane preparations the kinetics of 125I-IL1 binding were altered in the presence of guanosine 5'-[beta gamma-imido]triphosphate, resulting in the formation of a higher-affinity state for IL1 binding. Adenosine 5'-[beta gamma-imido]triphosphate at the same concentration was without effect. These results suggest that IL1 receptor function may be regulated by guanine nucleotides; however, the mechanism appears to differ from that exhibited by conventional G-protein-linked receptors. The lack of significant effects of IL1 on cAMP metabolism in these cells suggests that alternative pathways must exist to mediate the intracellular responses to stimulation via both types of the IL1 receptor.

Analysis of mutations in the putative nuclear localization sequence of interleukin-1 beta.

Previous studies have shown that, after receptor-mediated endocytosis, interleukin-1 alpha (IL1 alpha) and interleukin-1 beta (IL1 beta) are translocated to the nucleus, where they appear to accumulate. It has been suggested that nuclear translocation may be involved in the biological responsiveness of target cells to IL1 stimulation. The human IL1 beta molecule contains a seven-amino-acid sequence (-Pro208-Lys-Lys-Lys-Met-Glu-Lys-) that shows some sequence identity with the nuclear localization sequence of the simian-virus-40 large T-antigen. The effects of point mutations within this putative nuclear localization sequence on IL1 beta binding, receptor-mediated endocytosis and biological activity have been characterized. Mutants M49 (Lys210----Ala), M50 (Lys211----Ala) and M51 (Pro208----Ala) all retained the ability to bind to the IL1 receptor, albeit with lower affinity than the wild-type molecules. However, mutants M49, M50 and M51 showed greater biological potency than wild-type IL1 alpha or IL1 beta, as measured by the induction of IL2 secretion. However, receptor-mediated endocytosis and nuclear accumulation of M50 were comparable with those in the wild-type. These observations suggest that the putative nuclear localization sequence may play an important role in the generation of biological responses to IL1 stimulation, even though it may not influence internalization of the ligand.

Modification of biological responses to interleukin-1 by agents that perturb signal transduction pathways.

In this study we have examined the effect of agents known to perturb certain signal transduction pathways on the biological responses of target cells to stimulation with interleukin-1 (IL-1). In the murine thymoma cell line EL4, IL-1 stimulation results in the secretion of interleukin-2 (IL-2), which was subsequently measured by proliferation of an IL-2-dependent cell line. Agents that elevated intracellular cAMP blocked or partially blocked IL-1 induction of IL-2 secretion, whereas agents that activated protein kinase C (PKC) resulted in a synergistic enhancement. Both pertussis and cholera toxins also inhibited IL-1-induced IL-2 secretion, although probably by acting at different levels. IL-1 simulation of human and murine fibroblasts resulted in release of prostaglandin E2. This response was inhibitable by pertussis toxin but not by cholera toxin, whereas co-stimulation of the fibroblasts with IL-1 and phorbol ester resulted in a synergistic response. Murine fibroblasts could also be stimulated to proliferate by IL-1, and this response was also inhibitable by pertussis toxin. These findings are consistent with coupling of the IL-1 receptor to a signalling pathway via a pertussis toxin substrate.

Interleukin 1 responsiveness and receptor expression by murine TH1 and TH2 clones.

Murine Th1 and Th2 T cell lines differ in their responses to interleukin 1 (IL 1). Therefore, we examined two T-cell lines, D10.G4.1 (Th2) and MTg12B (Th1) in an attempt to correlate IL 1 receptor (IL 1R) expression with their IL 1 responsiveness. D10.G4.1 cells, which respond to IL 1, expressed two forms of the IL 1R, with molecular masses of approximately 80 kDa and approximately 60 kDa. In contrast, MTg12B cells failed to respond to IL 1 and only expressed the approximately 60 kDa receptor form. This suggests that the approximately 80 kDa receptor is essential for signaling. Expression of both IL 1R forms on D10.G4.1 cells could be inhibited by the anti-IL 4 antibody, 11B11. Antigen presentation reversibly upregulated both forms of the IL 1R, whereas stimulation with concanavalin A (ConA) and anti-CD3 only upregulated the approximately 60 kDa moiety. Upregulation of the approximately 80-kDa IL 1R by repeated antigenic stimulation resulted in a marked increase in sensitivity of D10.G4.1 cells to IL 1.

Receptor-mediated endocytosis and nuclear transport of human interleukin 1 alpha.

In this study we demonstrate that 125I-labelled interleukin (IL) 1 alpha binds specifically to its receptor on the surface of EL4 6.1 cells and is subsequently endocytosed and translocated from the cell membrane to the nucleus, where it progressively accumulates. Two-dimensional polyacrylamide-gel electrophoresis revealed that the internalized 125I-IL1 alpha associated with the nucleus was intact, with negligible breakdown products present. Specific and saturable binding of 125I-IL1 alpha was demonstrated on purified nuclei isolated from these cells. Binding of the radiolabelled ligand showed similar kinetics to that of the plasma-membrane receptor, and was inhibited by both unlabelled IL1 alpha and IL1 beta. Equilibrium binding studies on isolated nuclei revealed a single high-affinity binding site, with a Kd of 17 +/- 2 pM, and 79 +/- 12 binding sites per nucleus. These studies demonstrate that receptor-mediated endocytosis of IL1 results in its accumulation in the nucleus, and this mechanism may play an important role in mediating some of the actions of IL1.