Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model.

Airways inflammation is thought to play a central role in the pathogenesis of asthma. However, the precise role that individual inflammatory cells and mediators play in the development of airways hyperreactivity and the morphological changes of the lung during allergic pulmonary inflammation is unknown. In this investigation we have used a mouse model of allergic pulmonary inflammation and interleukin (IL) 5-deficient mice to establish the essential role of this cytokine and eosinophils in the initiation of aeroallergen-induced lung damage and the development of airways hyperreactivity. Sensitization and aerosol challenge of mice with ovalbumin results in airways eosinophilia and extensive lung damage analogous to that seen in asthma. Aeroallergen-challenged mice also display airways hyperreactivity to beta-methacholine. In IL-5-deficient mice, the eosinophilia, lung damage, and airways hyperreactivity normally resulting from aeroallergen challenge were abolished. Reconstitution of IL-5 production with recombinant vaccinia viruses engineered to express this factor completely restored aeroallergen-induced eosinophilia and airways dysfunction. These results indicate that IL-5 and eosinophils are central mediators in the pathogenesis of allergic lung disease.

Recombinant human interleukin 5 is a selective activator of human eosinophil function.

Human rIL-5 was found to selectively stimulate morphological changes and the function of human eosinophils. This molecule is thus a prime candidate for the selective eosinophilia and eosinophil activation seen in disease.

Relationship between interleukin-5 and eotaxin in regulating blood and tissue eosinophilia in mice.

The mechanism of cooperation between IL-5 and eotaxin for the selective accumulation of eosinophils at sites of allergic inflammation is unknown. In this investigation we have used IL-5 deficient mice to define the relationship between this cytokine and eotaxin in the regulation of blood eosinophilia and eosinophil homing and tissue accumulation. Both IL-5 and eotaxin could independently induce a rapid and pronounced blood eosinophilia in wild type mice when administered systemically. In contrast, only eotaxin induced a pronounced blood eosinophilia in IL-5 deficient mice. The eosinophilic response induced by intravenous eotaxin in wild type mice did not correlate with a significant reduction in the level of bone marrow eosinophils, whereas intravenous IL-5 resulted in depletion of this store. These results suggest the existence of two mechanisms by which eosinophils can be rapidly mobilized in response to intravenous eosinophil chemoattractants; first, mobilization of an IL-5 dependent bone marrow pool, and second, an eotaxin-induced sequestration of eosinophils from tissues into the blood. Subcutaneous injection of eotaxin induced a local tissue eosinophilia in wild type mice but not in IL-5 deficient mice. Furthermore, tissue eosinophilia in wild type mice, but not in IL-5 deficient mice, was enhanced by adoptive transfer of eosinophils or the administration of intravenous IL-5. However, pretreatment of IL-5 deficient mice with intraperitoneal IL-5 for 72 h restored eosinophil homing and tissue accumulation in response to subcutaneous eotaxin. We propose that eotaxin secreted from inflamed tissue may play an important role in initiating both blood and tissue eosinophilia in the early phases of allergic inflammation. Furthermore, IL-5 is not only essential for mobilizing eosinophils from the bone marrow during allergic inflammation, but also plays a critical role in regulating eosinophil homing and migration into tissues in response to eotaxin and possibly other specific chemotactic stimuli.

An improved resolution structure of the human beta common receptor involved in IL-3, IL-5 and GM-CSF signalling which gives better definition of the high-affinity binding epitope.

X-ray diffraction has been used to produce and refine a model of the extracellular domains of the beta common cytokine receptor. A minor improvement in resolution has resulted in improved electron-density maps, which have given a clearer indication of the position and stabilization of the key residues Tyr15, Phe79, Tyr347, His349, Ile350 and Tyr403 in the elbow region between domain 1 and domain 4 of the dimer-related molecule.

Correlation of interleukin-2 receptor expression with tissue-specific growth of an interleukin-3-dependent autocrine leukemia.

An autocrine leukemia (FDC-P1-IL3) has been developed using a retroviral vector containing the interleukin-3 (IL-3) gene to transfect the IL-3-dependent cell line FDC-P1. When leukemia cells were reisolated from experimental animals, it was found that levels of interleukin-2 (IL-2) receptor (IL-2R) expression were greater on cells isolated from the lymph node than on cells isolated from the spleen. Cloned sublines of FDC-P1-IL3 were selected by flow microfluorometry for high or low levels of IL-2R expression. Those clones that expressed high levels of IL-2R grew preferentially in the lymph node. Although IL-2 is not mitogenic for FDC-P1 cells and does not increase the rate of growth of FDC-P1-IL3 cells in vitro, the cloning efficiency of FDC-P1-IL3 is increased fourfold in the presence of IL-2. These observations suggest that the IL-2R on FDC-P1-IL3 cells plays an important role in modulating the growth of this leukemia in sites that contain high levels of IL-2.

Mutations affecting the reduced nicotinamide adenine dinucleotide dehydrogenase complex of Escherichia coli.

A strain carrying a point mutation affecting the NADH dehydrogenase complex of Escherichia coli has been isolated and its properties examined. The gene carrying the mutation (designated ndh) was located on the E. coli chromosome at about minute 23 and was shown to be cotransducible with the pyrC gene. Strain carrying the ndh- allele were found to be unable to grow on mannitol and to grow very poorly on glucose unless the medium was supplemented with succinate, acetate or casamino acids. The following properties of strains carrying the ndh- allele were established which suggest that the mutation affects the NADH dehydrogenase complex but apparently not the primary dehydrogenase. Membrane preparations possess normal to elevated levels of D-lactate oxidase and succinate oxidase activities but NADH oxidase is absent. NADH is unable to reduce ubiquinone in the aerobic steady state and reduces cytochrome b very slowly when the membranes become anaerobic. NADH dehydrogenase, measured as NADH-dichlorophenolindophenol reductase is reduced but not absent. NADH oxidase is stimulated by menadione although not by Q-3 or MK-1 and in the presence of menadione, cytochrome b is reduced normally by NADH. Further mutants affected in NADH oxidase were isolated using a screening procedure based on the growth characteristics of the original ndh- strain. The mutantions carried by these strains were all cotransducible with the pyrC gene and the biochemical properties of the additional mutants were similar to those of the original mutant. The properties of the group of ndh- mutants established so far suggest that they are affected in the transfer of reducing equivalents from the NADH dehydrogenase complex to ubiquinone.

Aerobic respiration in mutants of Escherichia coli accumulating quinone analogues of ubiquinone.

The ability of three naturally occurring analogues of ubiquinone to function in aerobic respiration in Escherichia coli has been studied. The compounds, which differ from ubiquinone in terms of the substituents on the quinone ring, accumulate in the cytoplasmic membranes of ubiE-, ubiF- and ubiG- mutants. One of the analogues (2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinone, NMQ), which lacks the 5-methoxyl group of the benzoquinone ring of ubiquinone promoted the oxidation of NADH, D-lactate and alpha-glycerophosphate but not succinate. Electron transport supported by MMQ was found to be coupled to phosphorylation. In contrast, 2-octaprenyl-6-methoxy-1,4-benzoquinone, which lacks both the 3-methyl and 5-methoxyl groups of ubiquinone, and 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone, in which the 5-methoxyl group of ubiquinone is replaced by an hydroxyl group, were virtually inactive in the oxidases tested. The ability of MMQ to function in respiration in isolated membranes is consistent with the findings that the growth rate and yield of a ubiF- strain, unlike other ubi- strains, were only slightly lower than those of a ubiF+ strain. The fact that MMQ is active in some but not all oxidases provides further support for the concept that the quinones link the individual dehydrogenases to the respiratory chain and that each dehydrogenase has specific structural requirements for quinone acceptors.

Role of quinones in electron transport to oxygen and nitrate in Escherichia coli. Studies with a ubiA- menA- double quinone mutant.

A ubiA- menA- double quinone mutant of Escherichia coli K12 was constructed together with other isogenic strains lacking either ubiquinone or menaquinone. These strains were used to study the role of quinones in electron transport to oxygen and nitrate. Each of the four oxidases examined (NADH, D-lactate, alpha-glycerophosphate and succinate) required a quinone for activity. Ubiquinone was active in each oxidase system while menaquinone gave full activity in alpha-glycerophosphate oxidase, partial activity in D-lactate oxidase but was inactive in NADH and succinate oxidation. The aerobic growth rates, growth yields and products of glucose metabolism of the quinone-deficient strains were also examined. The growth rate and growth yield of the ubi+menA- strain was the same as the wild-type strain, whereas the ubiA-men+ strain grew more slowly on glucose, had a lower growth yield (30% of wild type) and accumulated relatively large quantities of acetate and lactate. The growth of the ubiA-menA- strain was even more severely affected than that of the ubiA-men+ strain. Electron transport from formate, D-lactate, alpha-glycerophosphate and NADH to nitrate was also highly dependent on the presence of a quinone. Either ubiquinone or menaquinone was active in electron transport from formate and the activity of the quinones in electron transport from the other substrates was the same as for the oxidase systems. In contrast, quinones were not obligatory carriers in the anaerobic formate hydrogenlyase system. It is concluded that the quinones serve to link the various dehydrogenases with the terminal electron transport systems to oxygen and nitrate and that the dehydrogenases possess a degree of selectivity with respect to the quinone acceptors.

Biosynthesis of enterochelin in Escherichia coli K-12: separation of the polypeptides coded for by the entD, E, F and G genes.

Four enzymic components, coded for by the entD, entE, entF and entG genes, involved in the biosynthesis of enterochelin from 2,3-dihydroxybenzoate have been separated from cell extracts of mutant strains of Escherichia coli K-12. The starting material for fractionation of the E, F and G components was a cell extract of an entD mutant strain, which yielded the E, F and G enzymic components uncontaminated by a functional D component. The D component was isolated from cell extracts of an entE mutant strain. The conversion of 2,3-dihydroxybenzoate and L-serine into enterochelin is dependent on the presence of all four enzymic components. The E and F components were shown to catalyze ATP-pyrophosphate exchange reactions dependent on 2,3-dihydroxybenzoate and L-serine, respectively, whereas fractionated extracts of the entE and entF mutant strains lacked these reactions. These data provide firm evidence that the E and F components are involved in the initial activation of the substrates. The D and G components are necessary for subsequent and, as yet, undefinedd reactions.

Membrane-associated reactions in ubiquinone biosynthesis in Escherichia coli. 3-Octaprenyl-4-hydroxybenzoate carboxy-lyase.

A sensitive and quantitative assay for 3-octaprenyl-4-hydroxybenzoate carboxy-lyase has been developed. This enzyme, which catalyses the third reaction in ubiquinone biosynthesis in Escherichia coli, was partially purified and some of its properties determined. It was found that a considerable proportion of the carboxylyase activity could be separated from the membrane fraction in cell extracts prepared using a French press. Gel filtration showed the molecular weight of the enzyme to be about 340 000. For optimal activity the carboxy-lase was shown to require Mn2+, washed membranes or an extract of phospholipids, and an unidentified heat stable factor of molecular weight less than 10 000. The carboxy-lyase reaction was also shown to be strongly stimulated by dithiothreitol and methanol. The properties of the carboxy-lyase are compared with the three other enzymes concerned with ubiquinone biosynthesis in E. coli which have been studied in vitro. The fact that the substrate of the carboxy-lyase is membrane-bound and the enzyme is stimulated by phospholipid suggests that it normally functions in association with the cytoplasmic membrane in vivo.

Membrane-associated reactions in ubiquinone biosynthesis. 2-Octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone methyltransferase.

The O-methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone, which has been previously postulated to be the final reaction in the biosynthesis of ubiquinone was demonstrated in vitro using cell extracts of Escherichia coli. S-Adenosyl-L-methionine was active as the methyl donor for the reaction. The enzyme concerned, S-adenosyl-L-methionine: 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone-O-methyltransferase, was partially purified and shown to have a molecular weight of about 50 000 and to require a divalent metal and dithiothreitol for optimal activity in vitro. The methyltransferase was absent from extracts from ubiG- mutants suggesting that the ubiG gene is the structural gene coding for the methyltransferase. The enzyme, although not firmly membrane-bound, showed some affinity for the cell membrane in broken cell preparations and could utilize the benzoquinone substrate when the latter was free or bound to the cell membrane, with about equal efficiency. It is concluded that in vivo, the methyltransferase reaction probably occurs at the internal surface of the cytoplasmic membrane.

Comparison of the mechanisms regulating IL-5, IL-4, and three other lymphokine genes in the Th2 clone D10.G4.1.

The selective eosinophilia characteristic of asthma and parasite infections appears to be dependent on the production of interleukin-5 (IL-5) by activated T lymphocytes; however, little is known about IL-5 gene regulation. In the present study, a comparison was made of the basic mechanisms regulating mRNA levels for IL-5 and four other lymphokines that are coordinately expressed in the Th2 clone D10.G4.1 in response to concanavalin A (Con A) stimulation. Northern blot and nuclear run-off analyses indicated that IL-5 and IL-4 mRNA levels are primarily regulated at the transcriptional level. Regulation of IL-3, IL-6, and IL-10 mRNA levels involved control of mRNA stability and transcription. These three mRNAs were stabilized by cycloheximide (CHX). De novo protein synthesis was obligatory for IL-5 gene transcription and was also required for maximal transcription of the IL-4 and IL-10 genes. Expression of the IL-5, IL-6, and IL-10 genes was resistant to cyclosporine A (CsA), whereas IL-3 and IL-4 gene expression was completely inhibited, indicating the involvement of different signalling pathways in the regulation of these genes. The results indicate that the IL-5 gene and each of the other genes studied are independently regulated, providing the possibility of non-coordinate expression in vivo depending on the nature of the signals received during T cell activation.

Interleukin 1 plus interleukin 3 plus colony-stimulating factor 1 are essential for clonal proliferation of primitive myeloid bone marrow cells.

The clonal growth in nutrient agar at low cell densities of high-proliferative potential colony-forming cells (HPP-CFC) of bone marrow obtained from mice treated 2 days earlier with 5-fluorouracil (FU) (FU2dBM) has been shown to require a combination of three growth factors, interleukin 1 (IL-1), interleukin 3 (IL-3), and macrophage colony-stimulating factor (CSF-1). These HPP-CFC have been enriched 140-fold from FU2dBM by fluorescence-activated cell sorting of 7/4-, B220-, and L3T4-negative cells. The mean of the plating efficiencies of these enriched populations was 4.4% and no growth was observed when the factors were used singly. Similarly, enrichments of 16-fold were obtained from FU2dBM using immunomagnetic Dynabeads with anti-7/4 plus anti-B220 (meaning plating efficiency 0.5%). The further additions of human granulocyte CSF or mouse granulocyte-macrophage CSF or both to IL-1 plus IL-3 plus CSF-1 did not increase HPP-CFC colony formation, but both augmented the small colony formation with IL-1 plus IL-3, IL-3 plus CSF-1, or IL-1 plus CSF-1.

Conjugal transfer of cloning vectors derived from ColE1.

The transfer properties of five cloning vectors derived from ColE1 were studied. Two of the vectors (pSF2124 and pGM706) behaved like wild type ColE1 in that they could be transferred efficiently in the presence of the conjugative plasmid F. The mobilization of the remaining three vectors (pMB9, PBR313 and pBR322) by F was barely detectable. The transfer defect in pBR313 and pBR322 could be complemented by ColK when R64drd11, but not F, was used as the conjugative plasmid. The transferred plasmids could be recovered unchanged from recipients. Conjugal transfer is a potentially useful technique for screening hybrid plasmids in low-risk cloning experiments involving poorly transformable strains.

Tn5 mutagenesis of the enterochelin gene cluster of Escherichia coli.

Three recombinant plasmids have been studied which collectively carry all the nine known genes of the enterochelin system of iron transport in Escherichia coli K-12. The genetic organization of these genes was studied using a combination of Tn5 mutagenesis, subcloning of restriction fragments and restriction mapping. The order of the genes was established as entD, fes, entF, fep, and entCAGBE and the nine genes were shown to be distributed across 26 kb of DNA. The results of these studies indicate that the genes are organized into at least six transcriptional units.

The genetic code in bovine mitochondria: sequence of genes for the cytochrome oxidase subunit II and two tRNAs.

Bovine-heart mitochondrial DNA from a single animal was isolated and fragments representative of the entire genome cloned into multicopy plasmid vectors to facilitate determination of its complete nucleotide sequence. We present here the sequence of the region covering the gene for cytochrome oxidase subunit II. Comparison of this sequence with the amino acid sequence of the homologous beef-heart protein has enabled the determination of most of the bovine mitochondrial genetic code. The code differs from the "universal" genetic code in that UGA codes for tryptophan and not termination, and AUA codes for methionine and not isoleucine. The only codon family not represented is the AGA/AGG pair normally used for arginine; evidence from other genes suggests that these code for termination in bovine mitochondria. The sequence presented also includes the adjacent tRNAAsp and tRNALys genes. The tRNAAsp gene is separated by one nucleotide from the 5' end of the COII gene and only three bases separate the 3' end of this gene and the adjacent tRNALys gene. This highly compact gene organisation is very similar to that found in the corresponding region of the human mitochondrial genome and the gene arrangement is identical. The structure of the respective bovine and human tRNAs vary primarily the "D-" and "T psi C-loops".

Amplification of the respiratory NADH dehydrogenase of Escherichia coli by gene cloning.

A relatively simple method has been used to clone the gene coding for the respiratory NADH dehydrogenase (NADH-ubiquinone oxidoreductase) of Escherichia coli from unfractionated chromosomal DNA. The restriction endonucleases EcoRI, BamI and HindIII were used to construct three hybrid plasmid pools from total E. coli DNA and the amplifiable plasmids pSF2124 and pGM706. Three different restriction endonucleases were used to increase the chances of cloning the ndh gene intact. Mobilization by the plasmid F was used to transfer the hybrid plasmids into ndh mutants and selection was made for Apr and complementation of ndh. DNA fragments complementing ndh were isolated from both the EcoRI and HindIII hybrid plasmid pools. The strain carrying the hybrid plasmid constructed with EcoRI produced about 8--10 times the normal level of the respiratory NADH dehydrogenase in the cytoplasmic membrane. Treating the cells with chloramphenicol to increase the plasmid copy number allowed the level of NADH dehydrogenase in the membrane to be increased to 50--60 times the level in the wild type. The results indicate the potential of gene cloning for the specific amplification of particular proteins prior to their purification.

Bacteriophage Mu-mediated gene transposition and in vitro cloning of the enterochelin gene cluster of Escherichia coli.

Transposition of chromosomal genes using bacteriophage Mu has been used to obtain a partial order of the nine closely linked genes of the enterochelin-dependent iron transport system of Escherichia coli K-12. Fragments of the ent gene cluster were transposed into the conjugative plasmid RP4 and were characterized by genetic complementation. The partial gene order (entD, fes), entF, fep, entC, ent(ABEG)...lip was derived using six plasmids which carried overlapping parts of the cluster, and the fep mutations were shown to belong to a single complementation group. Two restriction fragments, one carrying ent(ABCEG) and the other carrying fep, were cloned in vitro using one of the RP4::ent plasmids as a source of DNA enriched in enterochelin system genes. A further restriction fragment, carrying the three remaining genes, entD, fes and entF was cloned directly from the chromosome. The three restriction fragments collectively cover a region of the chromosome 29 kg in length, indicating that the genes of the enterochelin system are clustered but not contiguous.

A functional bipartite nuclear localisation signal in the cytokine interleukin-5.

Interleukin (IL)-5 is central in regulating eosinophilia in allergic disease and parasitic infections. We have identified a bipartite nuclear localisation signal (NLS) within amino acids 95-111 of human IL-5 (hIL-5), also present in mouse IL-5 (mIL-5). hIL-5 and mIL-5 were labelled fluorescently, and nuclear uptake subsequent to membrane binding and internalisation by intact receptor expressing cells visualised and quantified using confocal laser scanning microscopy. hIL-5 and mIL-5 were shown to be transported to the nucleus in in vivo and in vitro nuclear protein import assays. The hIL-5 NLS was able to target a heterologous protein to the nucleus both in vivo and in vitro. Mutations within the proximal arm of the NLS abrogated nuclear targeting activity, confirming its bipartite nature. The results imply a nuclear signalling role for IL-5 additional to pathways linked to the membrane receptor system.

The cytokine interleukin-5 (IL-5) effects cotransport of its receptor subunits to the nucleus in vitro.

Interleukin (IL)-5 is central in regulating eosinophilia in allergic disease and parasitic infections. We have recently shown that human (h) IL-5 both possesses a functional nuclear localization signal capable of targeting a heterologous protein to the nucleus and localises to the nucleus of intact receptor-expressing cells. In this study, the extracellular domains of the hIL-5 alpha- and beta-receptor subunits were expressed in baculovirus, fluorescently labelled and assayed for nuclear targeting in vitro in the absence and presence of IL-5. The beta-subunit, which lacks IL-5 binding activity, only accumulated in the nucleus in the presence of both the hIL-5 binding alpha-subunit and hIL-5. The IL-5-binding alpha-subunit showed similar results. IL-5 thus effected nuclear transport of its alpha- and beta-receptor subunits apparently through a 'piggy back' mechanism, raising the possibility that IL-5's nuclear signalling role may be to cotarget its receptor subunits to the nucleus. This is the first demonstration of nuclear protein piggy back transport in vitro.