Identification of microRNAs regulating the developmental pathways of bone marrow derived mast cells.

BACKGROUND: MicroRNAs (miRNAs) play important roles in leukocyte differentiation, although those utilised for specific programs and key functions remain incompletely characterised. As a global approach to gain insights into the potential regulatory role of miRNA in mast cell differentiation we characterised expression in BM cultures from the initiation of differentiation. In cultures enriched in differentiating mast cells we characterised miRNA expression and identified miRNA targeting the mRNA of putative factors involved in differentiation pathways and cellular identity. Detailed pathway analysis identified a unique miRNA network that is intimately linked to the mast cell differentiation program. METHODOLOGY/PRINCIPAL FINDINGS: We identified 86 unique miRNAs with expression patterns that were up- or down- regulated at 5-fold or more during bone marrow derived mast cells (BMMC) development. By employing TargetScan and MeSH databases, we identified 524 transcripts involved in 30 canonical pathways as potentially regulated by these specific 86 miRNAs. Furthermore, by applying miRanda and IPA analyses, we predict that 7 specific miRNAs of this group are directly associated with the expression of c-Kit and FcεRIα and likewise, that 18 miRNAs promote expression of Mitf, GATA1 and c/EBPα three core transcription factors that direct mast cell differentiation. Furthermore, we have identified 11 miRNAs that may regulate the expression of STATs-3, -5a/b, GATA2 and GATA3 during differentiation, along with 13 miRNAs that target transcripts encoding Ndst2, mMCP4 and mMCP6 and thus may regulate biosynthesis of mast cell secretory mediators. CONCLUSIONS/SIGNIFICANCE: This investigation characterises changes in miRNA expression in whole BM cultures during the differentiation of mast cells and predicts functional links between miRNAs and their target mRNAs for the regulation of development. This information provides an important resource for further investigations of the contributions of miRNAs to mast cell differentiation and function.

Expression profiling of differentiating eosinophils in bone marrow cultures predicts functional links between microRNAs and their target mRNAs.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that regulate complex transcriptional networks underpin immune responses. However, little is known about the specific miRNA networks that control differentiation of specific leukocyte subsets. In this study, we profiled miRNA expression during differentiation of eosinophils from bone marrow (BM) progenitors (bmEos), and correlated expression with potential mRNA targets involved in crucial regulatory functions. Profiling was performed on whole BM cultures to document the dynamic changes in miRNA expression in the BM microenvironment over the differentiation period. miRNA for network analysis were identified in BM cultures enriched in differentiating eosinophils, and chosen for their potential ability to target mRNA of factors that are known to play critical roles in eosinophil differentiation pathways or cell identify. METHODOLOGY/PRINCIPAL FINDINGS: We identified 68 miRNAs with expression patterns that were up- or down- regulated 5-fold or more during bmEos differentiation. By employing TargetScan and MeSH databases, we identified 348 transcripts involved in 30 canonical pathways as potentially regulated by these miRNAs. Furthermore, by applying miRanda and Ingenuity Pathways Analysis (IPA), we identified 13 specific miRNAs that are temporally associated with the expression of IL-5Rα and CCR3 and 14 miRNAs associated with the transcription factors GATA-1/2, PU.1 and C/EBPε. We have also identified 17 miRNAs that may regulate the expression of TLRs 4 and 13 during eosinophil differentiation, although we could identify no miRNAs targeting the prominent secretory effector, eosinophil major basic protein. CONCLUSIONS/SIGNIFICANCE: This is the first study to map changes in miRNA expression in whole BM cultures during the differentiation of eosinophils, and to predict functional links between miRNAs and their target mRNAs for the regulation of eosinophilopoiesis. Our findings provide an important resource that will promote the platform for further understanding of the role of these non-coding RNAs in the regulation of eosinophil differentiation and function.

High yield production of a soluble human interleukin-3 variant from E. coli with wild-type bioactivity and improved radiolabeling properties.

Human interleukin-3 (hIL-3) is a polypeptide growth factor that regulates the proliferation, differentiation, survival and function of hematopoietic progenitors and many mature blood cell lineages. Although recombinant hIL-3 is a widely used laboratory reagent in hematology, standard methods for its preparation, including those employed by commercial suppliers, remain arduous owing to a reliance on refolding insoluble protein expressed in E. coli. In addition, wild-type hIL-3 is a poor substrate for radio-iodination, which has been a long-standing hindrance to its use in receptor binding assays. To overcome these problems, we developed a method for expression of hIL-3 in E. coli as a soluble protein, with typical yields of >3mg of purified hIL-3 per litre of shaking microbial culture. Additionally, we introduced a non-native tyrosine residue into our hIL-3 analog, which allowed radio-iodination to high specific activities for receptor binding studies whilst not compromising bioactivity. The method presented herein provides a cost-effective and convenient route to milligram quantities of a hIL-3 analog with wild-type bioactivity that, unlike wild-type hIL­3, can be efficiently radio-iodinated for receptor binding studies.

The role of interchain heterodisulfide formation in activation of the human common beta and mouse betaIL-3 receptors.

The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common beta (betac) receptor is shared by the three cytokines and functions together with cytokine-specific alpha subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human betac (hbetac) and unidentified cysteine residues in the N-terminal domains of the alpha receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hbetac is part of the cytokine binding epitope of this receptor and that an IL-3Ralpha isoform lacking the N-terminal Ig-like domain (the "SP2" isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hbetac and the related mouse receptor, beta(IL-3), could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hbetac led to both a complete loss of high affinity binding with the human IL-3Ralpha SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, beta(IL-3), not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Ralpha SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hbetac and beta(IL-3) in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.

Two modes of beta-receptor recognition are mediated by distinct epitopes on mouse and human interleukin-3.

The cytokine interleukin-3 (IL-3) is a critical regulator of inflammation and immune responses in mammals. IL-3 exerts its effects on target cells via receptors comprising an IL-3-specific alpha-subunit and common beta-subunit (beta c; shared with IL-5 and granulocyte-macrophage colony-stimulating factor) or a beta-subunit that specifically binds IL-3 (beta(IL-3); present in mice but not humans). We recently identified two splice variants of the alpha-subunit of the IL-3 receptor (IL-3R alpha) that are relevant to hematopoietic progenitor cell differentiation or proliferation: the full length ("SP1" isoform) and a novel isoform (denoted "SP2") lacking the N-terminal Ig-like domain. Although our studies demonstrated that each mouse IL-3 (mIL-3) R alpha isoform can direct mIL-3 binding to two distinct sites on the beta(IL-3) subunit, it has remained unclear which residues in mIL-3 itself are critical to the two modes of beta(IL-3) recognition and whether the human IL-3R alpha SP1 and SP2 orthologs similarly instruct human IL-3 binding to two distinct sites on the human beta c subunit. Herein, we describe the identification of residues clustering around the highly conserved A-helix residue, Glu(23), in the mIL-3 A- and C-helices as critical for receptor binding and growth stimulation via the beta(IL-3) and mIL-3R alpha SP2 subunits, whereas an overlapping cluster was required for binding and activation of beta(IL-3) in the presence of mIL-3R alpha SP1. Similarly, our studies of human IL-3 indicate that two different modes of beta c binding are utilized in the presence of the hIL-3R alpha SP1 or SP2 isoforms, suggesting a possible conserved mechanism by which the relative orientations of receptor subunits are modulated to achieve distinct signaling outcomes.

The Ig-like domain of human GM-CSF receptor alpha plays a critical role in cytokine binding and receptor activation.

GM-CSF (granulocyte/macrophage colony-stimulating factor) is an important mediator of inducible haemopoiesis and inflammation, and has a critical role in the function of alveolar macrophages. Its clinical applications include the mobilization of haemopoietic progenitors, and a role as an immune stimulant and vaccine adjuvant in cancer patients. GM-CSF signals via a specific alpha receptor (GM-CSFRalpha) and the shared hbetac (human common beta-subunit). The present study has investigated the role of the Ig-like domain of GM-CSFRalpha in GM-CSF binding and signalling. Deletion of the Ig-like domain abolished direct GM-CSF binding and decreased growth signalling in the presence of hbetac. To locate the specific residues in the Ig-like domain of GM-CSFRalpha involved in GM-CSF binding, a structural alignment was made with a related receptor, IL-13Ralpha1 (interleukin-13 receptor alpha1), whose structure and mode of interaction with its ligand has recently been elucidated. Mutagenesis of candidate residues in the predicted region of interaction identified Val51 and Cys60 as having critical roles in binding to the alpha receptor, with Arg54 and Leu55 also being important. High-affinity binding in the presence of hbetac was strongly affected by mutation of Cys60 and was also reduced by mutation of Val51, Arg54 and Leu55. Of the four key residues, growth signalling was most severely affected by mutation of Cys60. The results indicate a previously unrecognized role for the Ig-like domain, and in particular Cys60, of GM-CSFRalpha in the binding of GM-CSF and subsequent activation of cellular signalling.

A new isoform of interleukin-3 receptor {alpha} with novel differentiation activity and high affinity binding mode.

Interleukin-3 (IL-3) promotes both self-renewal and differentiation of early multipotential progenitors and is involved in inducible hematopoiesis in response to infections. Here we report new insights into these processes with the identification of a new isoform (SP2) of IL-3 receptor alpha (IL-3Ralpha), present in mouse and human hematopoietic cells, which lacks domain 1 of the full-length receptor (SP1). Binding assays with beta(IL-3) mutants showed that mouse SP2 uses a different high affinity binding mode to SP1, although both mouse and human SP2 and SP1 can stimulate IL-3-dependent growth. In IL-3-dependent differentiation models, human SP2 and SP1 gave differential effects on lineage commitment or self-renewal dependent on the cellular context, suggesting that different modes of ectodomain binding may modulate intracellular signaling. In a multipotential factor dependent cell-Paterson mix, the transcription factors C/EBPalpha and PU.1 and microRNAs miRNA-15a, -223, and -181a were up-regulated in cells undergoing SP2-supported differentiation compared with SP1-supported self-renewal. Similarly in M1 cells, SP2 promoted differentiation compared with SP1 and gave up-regulation of PU.1 and miRNA-155 and -223. These findings suggest that IL-3-promoted lineage commitment uses similar mechanisms to those of steady-state hematopoiesis. Both the SP1 and SP2 isoforms activated the Jak2/STAT5, Akt, and Erk1/2 signaling pathways in M1 cells, although the activation was more prolonged for the SP2 isoform.

Expression and evolution of the mammalian brain gene Ttyh1.

Homologues of the Drosophila melanogaster tweety (tty) gene are present in mammals and Caenorhabditis elegans. The encoded proteins have five predicted membrane-spanning regions and recent findings suggest that some family members may be chloride channels. Phylogenetic analysis of the tty family including novel members from slime mould Entamoeba and plants has revealed the occurrence of independent gene duplication events in different lineages. expressed sequence tag data indicate that expression of the mammalian Ttyh1 gene is restricted mainly to neural tissue and is up-regulated in astrocytoma, glioma and several other cancers. In this study, mammalian expression vectors were used to investigate the subcellular localization and the effect of over-expression of Ttyh1 in human epithelial kidney cells. The results confirm that Ttyh1 is a membrane protein and show that it is deposited on the substratum along the migration paths of motile cells above the alpha5beta1-integrin complex. The ectopic expression of Ttyh1 also induced long filopodia, which were branched and dynamic in both stationary and migratory cells. The filopodia contained F-actin and occurred at the ends of microtubules which were polarized towards the membrane. Upon contact with nearby cells some filopodia stabilized and filled with F-actin, whereas Ttyh1 was highly concentrated at the cell-cell interface. Ttyh1 N- and C-terminal antipeptide antibodies detected Ttyh1 along the axons of neurones in primary rat hippocampal cell cultures, and in situ in whole rat brain slices around the hippocampus and occasionally between cells. These data suggest a role for Ttyh1 in process formation, cell adhesion and possibly as a transmembrane receptor.

IL-3, IL-5, and GM-CSF signaling: crystal structure of the human beta-common receptor.

The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony stimulating factor (GM-CSF), are polypeptide growth factors that exhibit overlapping activities in the regulation of hematopoietic cells. They appear to be primarily involved in inducible hematopoiesis in response to infections and are involved in the pathogenesis of allergic and inflammatory diseases and possibly in leukemia. The X-ray structure of the beta common (betac) receptor ectodomain has given new insights into the structural biology of signaling by IL-3, IL-5, and GM-CSF. This receptor is shared between the three ligands and functions together with three ligand-specific alpha-subunits. The structure shows betac is an intertwined homodimer in which each chain contains four domains with approximate fibronectin type-III topology. The two betac-subunits that compose the homodimer are interlocked by virtue of the swapping of beta-strands between domain 1 of one subunit and domain 3 of the other subunit. Site-directed mutagenesis has shown that the interface between domains 1 and 4 in this unique structure forms the functional epitope. This epitope is similar to those of other members of the cytokine class I receptor family but is novel in that it is formed by two different receptor chains. The chapter also reviews knowledge on the closely related mouse beta(IL-3) receptor and on the alpha-subunit-ligand interactions. The knowledge on the two beta receptors is placed in context with advances in understanding of the structural biology of other members of the cytokine class I receptor family.

A role for Ets1, synergizing with AP-1 and GATA-3 in the regulation of IL-5 transcription in mouse Th2 lymphocytes.

IL-5 is a key regulator of eosinophilic inflammation and is selectively expressed by antigen-activated Th2 lymphocytes. An important role for the proximal AP-1 and GATA sites in regulating IL-5 transcription is generally accepted but the significance of an adjacent Ets/NFAT site has remained unclear. We have investigated its role using the mouse Th2 clone D10.G4.1. Transcription of IL-5 reporter gene plasmids could be induced in D10 cells by phorbol myristate acetate/cyclic adenosine monophosphate (PMA/cAMP) stimulation and significantly further enhanced by activation of the mitogen-activated protein (MAP) kinase pathways. Strong induction of IL-5 mRNA was also induced by PMA/cAMP. Mutagenesis showed that the Ets/NFAT site is of critical importance along with the AP-1 and GATA sites in regulating IL-5 transcription stimulated by PMA/cAMP and MAP kinase activation. Transactivation was used to investigate the transcription factors which could function at the three sites and possible synergistic interactions. AP-1 (c-Fos/c-Jun) strongly induced IL-5 transcription and dominant negative AP-1 constructs confirmed that AP-1 plays an important role in regulating IL-5 expression. Ets1, unlike other members of the Ets/NFAT family, synergized strongly with AP-1 suggesting that Ets1 is the family member which functions at the Ets/NFAT site. AP-1/Ets1 transactivation also stimulated IL-5 mRNA expression. Ets1 binding to the proximal promoter region, demonstrated by chromatin immunoprecipitation, was stimulated by PMA/cAMP. The absolute dependence on the binding sites for Ets1, AP-1 and GATA-3 together with the strong synergy between Ets1 and AP-1 suggest close cooperative interactions between the three transcription factors in the regulation of IL-5 expression in mouse T cells.

Interleukin-3 binding to the murine betaIL-3 and human betac receptors involves functional epitopes formed by domains 1 and 4 of different protein chains.

Interleukin-3 (IL-3) is a cytokine produced by activated T-cells and mast cells that is active on a broad range of hematopoietic cells and in the nervous system and appears to be important in several chronic inflammatory diseases. In this study, alanine substitutions were used to investigate the role of residues of the human beta-common (hbetac) receptor and the murine IL-3-specific (beta(IL-3)) receptor in IL-3 binding. We show that the domain 1 residues, Tyr(15) and Phe(79), of the hbetac receptor are important for high affinity IL-3 binding and receptor activation as shown previously for the related cytokines, interleukin-5 and granulocyte-macrophage colony-stimulating factor, which also signal through this receptor subunit. From the x-ray structure of hbetac, it is clear that the domain 1 residues cooperate with domain 4 residues to form a novel ligand-binding interface involving the two protein chains of the intertwined homodimer receptor. We demonstrate by ultracentrifugation that the beta(IL-3) receptor is also a homodimer. Its high sequence homology with hbetac suggests that their structures are homologous, and we identified an analogous binding interface in beta(IL-3) for direct IL-3 binding to the high affinity binding site in hbetac. Tyr(21) (A-B loop), Phe(85), and Asn(87) (E-F loop) of domain 1; Ile(320) of the interdomain loop; and Tyr(348) (B'-C' loop) and Tyr(401) (F'-G' loop) of domain 4 were shown to have critical individual roles and Arg(84) and Tyr(317) major secondary roles in direct murine IL-3 binding to the beta(IL-3)receptor. Most surprising, none of the key residues for direct IL-3 binding were critical for high affinity binding in the presence of the murine IL-3 alpha receptor, indicating a fundamentally different mechanism of high affinity binding to that used by hbetac.

A novel functional epitope formed by domains 1 and 4 of the human common beta-subunit is involved in receptor activation by granulocyte macrophage colony-stimulating factor and interleukin 5.

The receptors for human interleukins 3 and 5 and granulocyte macrophage colony-stimulating factor are composed of ligand-specific alpha-subunits and a common beta-subunit (betac), the major signaling entity. The way in which betac interacts with ligands in the respective activation complexes has remained poorly understood. The recently determined crystal structure of the extracellular domain of betac revealed a possible ligand-binding interface composed of domain 1 of one chain of the betac dimer and the adjacent domain 4 of the symmetry-related chain. We have used site-directed mutagenesis, in conjunction with ligand binding and proliferation studies, to demonstrate the critical requirement of the domain 1 residues, Tyr(15) (A-B loop) and Phe(79) (E-F loop), in high affinity complex formation and receptor activation. The novel ligand-receptor interface formed between domains 1 and 4 represents the first example of a class I cytokine receptor interface to be composed of two noncontiguous fibronectin III domains.

Interleukin-4 and interleukin-5 as targets for inhibition of eosinophilic inflammation and allergic airways hyperreactivity

Clinical and experimental investigations suggest that allergen-specific CD4+ T-cells, IgE and the cytokines IL-4 and IL-5 play central roles in initiating and sustaining an asthmatic response by regulating the recruitment and/or activation of airways mast cells and eosinophils. IL-5 plays a unique role in eosinophil development and activation and has been strongly implicated in the aetiology of asthma. The present paper summarizes our recent investigations on the role of these cytokines using cytokines knockout mice and a mouse aeroallergen model. Investigations in IL-5-/- mice indicate that this cytokines is critical for regulating aeroallergen-induced eosinophilia, the onset of lung damage and airways hyperreactivity during allergic airways inflammation. While IL-4 and allergen-specific IgE play important roles in the regulation of allergic disease, recent investigations in IL-4-/- mice suggest that allergic airways inflammation can occur via pathways which operate independently of these molecules. Activation of these IL-4 independent pathways are also intimately associated with CDA+ T-cells, IL-5 signal transduction and eosinophilic inflammation. Such IL-5 regulated pathways may also play a substantive role in the aetiology of asthma. Thus, evidence is now emerging that allergic airways disease is regulated by humoral and cell mediated pocesses. The central role of IL-5 in both components of allergic disease highlights the requirements for highly specific therapeutic agents which inhibit the production or action of this cytokines.

The role of interleukin-5 (IL-5) in vivo: studies with IL-5 deficient mice

Eosinophil recruitment is a characteristic feature of a number of pathological conditions and was the topic of the recent International Symposium on allergic inflammation, asthma, parasitic and infectious diseases (Rio de Janeiro, June 3-5, 1996). Since interleukin-5 (IL-5) is believed to regulate the gowth, differentiation and activation of eosinophils (Conffman et al. 1989, Sanderson 1992), the role of eosinophils and IL-5 are closely linked. Although IL-5 specifically regulates eosinophilia in vivo and this is its most well established activity, it is becoming clear that IL-5 also has other biological effects. The recent derivation of an IL-5 deficient mouse (Kopf et al. 1996), provides a model for exploring not only the role of IL-5 and eosinophils but also other novel activities of IL-5. Of note is that although the IL-5 deficient mice cannot elicit a pronounced eosinophilia in response of inflammatory stimulation following aeroallergen challenge or parasite infection they still produce basal levels of eosinophils that appear to be morphologically and functionally normal. However, the basal levels of eosinophils appear insufficient for normal host defense as IL-5 deficiency has been shown to compromise defence against several helminth infections. In addition, IL-5 deficient mice appear to have functional deficiencies in B-1 B lymphocytes and in IgA production.