A molecular switch changes the signalling pathway used by the FcγRI antibody receptor to mobilise calcium
Identifieur interne : 002654 ( Istex/Corpus ); précédent : 002653; suivant : 002655A molecular switch changes the signalling pathway used by the FcγRI antibody receptor to mobilise calcium
Auteurs : Alirio Melendez ; R. Andres Floto ; Angus J. Cameron ; David J. Gillooly ; Margaret M. Harnett ; Janet M. AllenSource :
- Current Biology [ 0960-9822 ] ; 1998.
English descriptors
- Teeft :
- Accessory, Accessory molecules, Activates, Activation, Aggregated, Aggregation, Antisense, Antisense chain, Antisense control oligonucleotide, Antisense oligonucleotides, Assay, Biol, Biol chem, Butanol, Calcium, Calcium oscillations, Calcium response, Calcium transients, Cell calcium, Chem, Control values, Current biology, Cytoplasmic tail, Dbcamp, Different phospholipid, Differentiation state, Distinct phospholipase pathways melendez, Fcgri, Harnett, High affinity, Human igg4, Immune, Immune complexes, Immunol, Inositol, Inositol phosphates, Insp3, Insp3 levels, Intracellular, Kinase, Ligand, Loading cells, Macrophage, Melendez, Molecular switch, Monoclonal, Monoclonal antibodies, Monoclonal antibody, Monocyte, Monocytic, Monocytic cell line, More details, Mrna, Myeloid cells, Oligonucleotide, Oligonucleotide antisense, Pathway, Phagocytosis, Phosphate, Phospholipase, Phospholipid, Phosphorylation, Proc natl acad, Protein kinase, Receptor, Receptor aggregation, Research paper fcgri activates, Separate experiments, Signal transduction, Single spike, Specific aggregation, Specific receptor, Sphingosine, Sphingosine kinase, Sphingosine kinase activation, Sphingosine kinase activity, Standard deviation, Supplementary material, Surface expression, Transduction, Transphosphatidylation assay, Triplicate, Triplicate measurements, Tyrosine kinases, Tyrosine phosphorylation.
Abstract
Abstract: Background: Leukocytes express Fcγ receptors, which are specific for the constant region of immunoglobulin G. Aggregation of these receptors activates a repertoire of responses that can lead to targeted cell killing by antibody directed cellular cytotoxicity. The nature of the myeloid response to Fcγ receptor aggregation is highly variable and depends on the maturation state of the cell, but little is known about the signalling mechanisms underlying this variability.Results: We show here that differentiation of a monocytic cell line, U937, to a more macrophage phenotype resulted in an absolute and fundamental switch in the nature of the phospholipid signalling pathway recruited following Fcγ receptor aggregation. In cytokine-primed monocytes, aggregation of the high-affinity receptor FcγRI resulted in the activation of phospholipase D and sphingosine kinase, which in turn led to the transient release of stored calcium; these effects were mediated by the γ chain, an FcγRI accessory protein. In contrast, in cells differentiated to a more macrophage type, aggregation of FcγRI resulted in the FcγRIIa-mediated activation of phospholipase C, and the resulting calcium response was prolonged as calcium entry was stimulated.Conclusions: The switch in FcγRI signalling pathways upon monocyte differentiation is mediated by a switch in the accessory molecule recruited by FcγRI, which lacks its own intrinsic signal transduction motif. As many immune receptors have separate polypeptide chains for ligand binding and signal transduction (allowing a similar switch in signalling pathways), the mechanism described here is likely to be widely used.
Url:
DOI: 10.1016/S0960-9822(98)70085-5
Links to Exploration step
ISTEX:B24ED7113398FAB8F911958DF2F198CFB7B64BD9Le document en format XML
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Allen</name><affiliation><mods:affiliation>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</mods:affiliation></affiliation><affiliation><mods:affiliation>Corresponding author.</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: janet.allen@bio.gla.ac.uk</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:B24ED7113398FAB8F911958DF2F198CFB7B64BD9</idno><date when="1996" year="1996">1996</date><idno type="doi">10.1016/S0960-9822(98)70085-5</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-TKRZZVTD-T/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">002654</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002654</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">A molecular switch changes the signalling pathway used by the FcγRI antibody receptor to mobilise calcium</title><author><name sortKey="Melendez, Alirio" sort="Melendez, Alirio" uniqKey="Melendez A" first="Alirio" last="Melendez">Alirio Melendez</name><affiliation><mods:affiliation>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</mods:affiliation></affiliation></author><author><name sortKey="Floto, R Andres" sort="Floto, R Andres" uniqKey="Floto R" first="R. Andres" last="Floto">R. Andres Floto</name><affiliation><mods:affiliation>Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG, UK</mods:affiliation></affiliation></author><author><name sortKey="Cameron, Angus J" sort="Cameron, Angus J" uniqKey="Cameron A" first="Angus J." last="Cameron">Angus J. Cameron</name><affiliation><mods:affiliation>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</mods:affiliation></affiliation></author><author><name sortKey="Gillooly, David J" sort="Gillooly, David J" uniqKey="Gillooly D" first="David J." last="Gillooly">David J. Gillooly</name><affiliation><mods:affiliation>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</mods:affiliation></affiliation></author><author><name sortKey="Harnett, Margaret M" sort="Harnett, Margaret M" uniqKey="Harnett M" first="Margaret M." last="Harnett">Margaret M. Harnett</name><affiliation><mods:affiliation>Department of Immunology, University of Glasgow, Glasgow G11 6NT, Scotland, UK</mods:affiliation></affiliation></author><author><name sortKey="Allen, Janet M" sort="Allen, Janet M" uniqKey="Allen J" first="Janet M." last="Allen">Janet M. Allen</name><affiliation><mods:affiliation>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</mods:affiliation></affiliation><affiliation><mods:affiliation>Corresponding author.</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: janet.allen@bio.gla.ac.uk</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Current Biology</title><title level="j" type="abbrev">CURBIO</title><idno type="ISSN">0960-9822</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1998">1998</date><biblScope unit="volume">8</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="210">210</biblScope><biblScope unit="page" to="222">222</biblScope></imprint><idno type="ISSN">0960-9822</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0960-9822</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Accessory</term><term>Accessory molecules</term><term>Activates</term><term>Activation</term><term>Aggregated</term><term>Aggregation</term><term>Antisense</term><term>Antisense chain</term><term>Antisense control oligonucleotide</term><term>Antisense oligonucleotides</term><term>Assay</term><term>Biol</term><term>Biol chem</term><term>Butanol</term><term>Calcium</term><term>Calcium oscillations</term><term>Calcium response</term><term>Calcium transients</term><term>Cell calcium</term><term>Chem</term><term>Control values</term><term>Current biology</term><term>Cytoplasmic tail</term><term>Dbcamp</term><term>Different phospholipid</term><term>Differentiation state</term><term>Distinct phospholipase pathways melendez</term><term>Fcgri</term><term>Harnett</term><term>High affinity</term><term>Human igg4</term><term>Immune</term><term>Immune complexes</term><term>Immunol</term><term>Inositol</term><term>Inositol phosphates</term><term>Insp3</term><term>Insp3 levels</term><term>Intracellular</term><term>Kinase</term><term>Ligand</term><term>Loading cells</term><term>Macrophage</term><term>Melendez</term><term>Molecular switch</term><term>Monoclonal</term><term>Monoclonal antibodies</term><term>Monoclonal antibody</term><term>Monocyte</term><term>Monocytic</term><term>Monocytic cell line</term><term>More details</term><term>Mrna</term><term>Myeloid cells</term><term>Oligonucleotide</term><term>Oligonucleotide antisense</term><term>Pathway</term><term>Phagocytosis</term><term>Phosphate</term><term>Phospholipase</term><term>Phospholipid</term><term>Phosphorylation</term><term>Proc natl acad</term><term>Protein kinase</term><term>Receptor</term><term>Receptor aggregation</term><term>Research paper fcgri activates</term><term>Separate experiments</term><term>Signal transduction</term><term>Single spike</term><term>Specific aggregation</term><term>Specific receptor</term><term>Sphingosine</term><term>Sphingosine kinase</term><term>Sphingosine kinase activation</term><term>Sphingosine kinase activity</term><term>Standard deviation</term><term>Supplementary material</term><term>Surface expression</term><term>Transduction</term><term>Transphosphatidylation assay</term><term>Triplicate</term><term>Triplicate measurements</term><term>Tyrosine kinases</term><term>Tyrosine phosphorylation</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: Background: Leukocytes express Fcγ receptors, which are specific for the constant region of immunoglobulin G. Aggregation of these receptors activates a repertoire of responses that can lead to targeted cell killing by antibody directed cellular cytotoxicity. The nature of the myeloid response to Fcγ receptor aggregation is highly variable and depends on the maturation state of the cell, but little is known about the signalling mechanisms underlying this variability.Results: We show here that differentiation of a monocytic cell line, U937, to a more macrophage phenotype resulted in an absolute and fundamental switch in the nature of the phospholipid signalling pathway recruited following Fcγ receptor aggregation. In cytokine-primed monocytes, aggregation of the high-affinity receptor FcγRI resulted in the activation of phospholipase D and sphingosine kinase, which in turn led to the transient release of stored calcium; these effects were mediated by the γ chain, an FcγRI accessory protein. In contrast, in cells differentiated to a more macrophage type, aggregation of FcγRI resulted in the FcγRIIa-mediated activation of phospholipase C, and the resulting calcium response was prolonged as calcium entry was stimulated.Conclusions: The switch in FcγRI signalling pathways upon monocyte differentiation is mediated by a switch in the accessory molecule recruited by FcγRI, which lacks its own intrinsic signal transduction motif. As many immune receptors have separate polypeptide chains for ligand binding and signal transduction (allowing a similar switch in signalling pathways), the mechanism described here is likely to be widely used.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>receptor</json:string><json:string>pathway</json:string><json:string>kinase</json:string><json:string>dbcamp</json:string><json:string>sphingosine</json:string><json:string>inositol</json:string><json:string>antisense</json:string><json:string>aggregation</json:string><json:string>inositol phosphates</json:string><json:string>monoclonal</json:string><json:string>receptor aggregation</json:string><json:string>monoclonal antibodies</json:string><json:string>phospholipase</json:string><json:string>sphingosine kinase</json:string><json:string>phospholipid</json:string><json:string>fcgri</json:string><json:string>insp3</json:string><json:string>specific aggregation</json:string><json:string>immune</json:string><json:string>monocyte</json:string><json:string>oligonucleotide</json:string><json:string>current biology</json:string><json:string>transduction</json:string><json:string>macrophage</json:string><json:string>immunol</json:string><json:string>butanol</json:string><json:string>aggregated</json:string><json:string>triplicate</json:string><json:string>activates</json:string><json:string>standard deviation</json:string><json:string>calcium response</json:string><json:string>signal transduction</json:string><json:string>triplicate measurements</json:string><json:string>biol</json:string><json:string>mrna</json:string><json:string>intracellular</json:string><json:string>melendez</json:string><json:string>calcium oscillations</json:string><json:string>monocytic</json:string><json:string>harnett</json:string><json:string>separate experiments</json:string><json:string>phosphorylation</json:string><json:string>immune complexes</json:string><json:string>biol chem</json:string><json:string>phagocytosis</json:string><json:string>chem</json:string><json:string>distinct phospholipase pathways melendez</json:string><json:string>ligand</json:string><json:string>antisense oligonucleotides</json:string><json:string>sphingosine kinase activity</json:string><json:string>tyrosine kinases</json:string><json:string>specific receptor</json:string><json:string>accessory</json:string><json:string>research paper fcgri activates</json:string><json:string>sphingosine kinase activation</json:string><json:string>antisense chain</json:string><json:string>monoclonal antibody</json:string><json:string>accessory molecules</json:string><json:string>high affinity</json:string><json:string>human igg4</json:string><json:string>protein kinase</json:string><json:string>proc natl acad</json:string><json:string>different phospholipid</json:string><json:string>assay</json:string><json:string>activation</json:string><json:string>phosphate</json:string><json:string>surface expression</json:string><json:string>tyrosine phosphorylation</json:string><json:string>transphosphatidylation assay</json:string><json:string>single spike</json:string><json:string>loading cells</json:string><json:string>differentiation state</json:string><json:string>molecular switch</json:string><json:string>monocytic cell line</json:string><json:string>antisense control oligonucleotide</json:string><json:string>control values</json:string><json:string>insp3 levels</json:string><json:string>myeloid cells</json:string><json:string>supplementary material</json:string><json:string>calcium transients</json:string><json:string>cytoplasmic tail</json:string><json:string>more details</json:string><json:string>cell calcium</json:string><json:string>oligonucleotide antisense</json:string><json:string>calcium</json:string></teeft></keywords><author><json:item><name>Alirio Melendez</name><affiliations><json:string>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</json:string></affiliations></json:item><json:item><name>R.Andres Floto</name><affiliations><json:string>Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG, UK</json:string></affiliations></json:item><json:item><name>Angus J. Cameron</name><affiliations><json:string>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</json:string></affiliations></json:item><json:item><name>David J. Gillooly</name><affiliations><json:string>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</json:string></affiliations></json:item><json:item><name>Margaret M. Harnett</name><affiliations><json:string>Department of Immunology, University of Glasgow, Glasgow G11 6NT, Scotland, UK</json:string></affiliations></json:item><json:item><name>Janet M. Allen</name><affiliations><json:string>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</json:string><json:string>Corresponding author.</json:string><json:string>E-mail: janet.allen@bio.gla.ac.uk</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Research Paper</value></json:item></subject><arkIstex>ark:/67375/6H6-TKRZZVTD-T</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: Background: Leukocytes express Fcγ receptors, which are specific for the constant region of immunoglobulin G. Aggregation of these receptors activates a repertoire of responses that can lead to targeted cell killing by antibody directed cellular cytotoxicity. The nature of the myeloid response to Fcγ receptor aggregation is highly variable and depends on the maturation state of the cell, but little is known about the signalling mechanisms underlying this variability.Results: We show here that differentiation of a monocytic cell line, U937, to a more macrophage phenotype resulted in an absolute and fundamental switch in the nature of the phospholipid signalling pathway recruited following Fcγ receptor aggregation. In cytokine-primed monocytes, aggregation of the high-affinity receptor FcγRI resulted in the activation of phospholipase D and sphingosine kinase, which in turn led to the transient release of stored calcium; these effects were mediated by the γ chain, an FcγRI accessory protein. In contrast, in cells differentiated to a more macrophage type, aggregation of FcγRI resulted in the FcγRIIa-mediated activation of phospholipase C, and the resulting calcium response was prolonged as calcium entry was stimulated.Conclusions: The switch in FcγRI signalling pathways upon monocyte differentiation is mediated by a switch in the accessory molecule recruited by FcγRI, which lacks its own intrinsic signal transduction motif. As many immune receptors have separate polypeptide chains for ligand binding and signal transduction (allowing a similar switch in signalling pathways), the mechanism described here is likely to be widely used.</abstract><qualityIndicators><score>9.892</score><pdfWordCount>7777</pdfWordCount><pdfCharCount>53232</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>13</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>241</abstractWordCount><abstractCharCount>1665</abstractCharCount><keywordCount>1</keywordCount></qualityIndicators><title>A molecular switch changes the signalling pathway used by the FcγRI antibody receptor to mobilise calcium</title><pmid><json:string>9501983</json:string></pmid><pii><json:string>S0960-9822(98)70085-5</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Current Biology</title><language><json:string>unknown</json:string></language><publicationDate>1998</publicationDate><issn><json:string>0960-9822</json:string></issn><pii><json:string>S0960-9822(00)X0239-4</json:string></pii><volume>8</volume><issue>4</issue><pages><first>210</first><last>222</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>18S</json:string><json:string>28S</json:string><json:string>1996</json:string></date><geogName><json:string>Fc region</json:string></geogName><orgName><json:string>Physiological Laboratory, University of Cambridge, Cambridge CB</json:string><json:string>Medical Research Council</json:string><json:string>PLD Research Paper</json:string><json:string>Current Biology Ltd</json:string><json:string>Bender Wein Ltd</json:string><json:string>University of Glasgow</json:string><json:string>Department of Medicine</json:string><json:string>Research Paper</json:string><json:string>Jandel Scientific Corp</json:string><json:string>Department of Immunology, University of Glasgow, Glasgow G</json:string><json:string>Division of Biochemistry</json:string><json:string>Cairn Research Spectrophotometer and cells</json:string><json:string>Wellcome Trust</json:string><json:string>MacFeat Bequest</json:string></orgName><orgName_funder><json:string>Wellcome Trust</json:string><json:string>MacFeat Bequest</json:string></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Janet M. Allen</json:string><json:string>Stewart Sage</json:string><json:string>G. Aggregation</json:string><json:string>Margaret M. Harnett</json:string><json:string>S. Seatter</json:string><json:string>The</json:string><json:string>T. McShane</json:string><json:string>David J. Gillooly</json:string><json:string>Angus J. Cameron</json:string></persName><placeName><json:string>UK</json:string></placeName><ref_url><json:string>http://biomednet.com/elecref/</json:string></ref_url><ref_bibl><json:string>[12,41]</json:string><json:string>[34]</json:string><json:string>[29]</json:string><json:string>[6]</json:string><json:string>Briscoe et al. [27]</json:string><json:string>[33]</json:string><json:string>Olivera et al. [40]</json:string><json:string>[8]</json:string><json:string>[39]</json:string><json:string>[14,15]</json:string><json:string>Melendez et al.</json:string><json:string>[32]</json:string><json:string>[8,10,11]</json:string><json:string>[38]</json:string><json:string>[35,36]</json:string><json:string>[20]</json:string><json:string>[37]</json:string><json:string>[12,13]</json:string><json:string>[7]</json:string><json:string>[7,19,33]</json:string><json:string>[14]</json:string><json:string>[24]</json:string><json:string>[27,28]</json:string><json:string>[4,5]</json:string><json:string>[19]</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-TKRZZVTD-T</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - cell biology</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - developmental biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Agricultural and Biological Sciences</json:string><json:string>3 - General Agricultural and Biological Sciences</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - General Biochemistry, Genetics and Molecular Biology</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>1998</publicationDate><copyrightDate>1996</copyrightDate><doi><json:string>10.1016/S0960-9822(98)70085-5</json:string></doi><id>B24ED7113398FAB8F911958DF2F198CFB7B64BD9</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-TKRZZVTD-T/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-TKRZZVTD-T/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-TKRZZVTD-T/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">A molecular switch changes the signalling pathway used by the FcγRI antibody receptor to mobilise calcium</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©1996 Elsevier Science Ltd</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>1996</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note type="content">Section title: Research Paper</note><note type="content">Figure 1: Differential generation of inositol phosphates following ligand activation of Fcγ receptor in dbc-AMP- and IFN-γ-treated cells. (a) InsP3 levels in dbc-AMP- or IFN-γ-treated cells at set times (30 sec to 20 min) after Fcγ receptor aggregation. In dbcAMP-differentiated cells, InsP3 concentrations were above control (samples with no added cross-linking antibody) values at all time points after Fcγ receptor aggregation, although concentrations appeared to oscillate. In IFN-γ-treated cells, InsP3 concentrations were never higher than control values at any time point. The data shown are the mean ± the standard deviation of triplicate measurements and are representative of four different experiments. (b) Total accumulated inositol phosphates following Fcγ receptor aggregation in dbcAMP- and IFN-γ-treated cells. Cells were treated with 10 mM lithium chloride. Cells were harvested at 5 min intervals after Fcγ receptor aggregation and assayed for inositol phosphate concentration. The data shown are the mean ± the standard deviation of triplicate measurements and are representative of five separate experiments.</note><note type="content">Figure 2: Relative DAG levels after aggregation of Fcγ receptors by ligand in dbcAMP- and IFN-γ-treated cells. The effect of butanol (0.3%) on the levels of DAG before (control) and after (X link) Fcγ receptor aggregation was examined. The data shown are the mean ± the standard deviation of triplicate measurements and are representative of four separate experiments.</note><note type="content">Figure 3: Measurement of PLD activity in dbcAMP-and IFN-γ-treated cells using the transphosphatidylation assay. (a) PLD activity, measured as the accumulation of [3H]PtdBut in cells before (control) and 30 min after (X link) aggregation of Fcγ receptors. (b) PLD activity in dbcAMP- or IFN-γ-treated cells following activation by phorbol ester (PMA). The data shown are the mean ± the standard deviation of triplicate measurements and are representative of six separate experiments.</note><note type="content">Figure 4: Response of dbcAMP-differentiated cells to specific cross-linking of FcγRI or FcγRIIa using monoclonal antibodies. (a,b) Accumulation of inositol phosphates at specified times after specific receptor cross-linking. (c,d) DAG generation 30 min after specific receptor cross-linking for the indicated times and the effect of butanol on DAG production. (e) PLD activity after specific receptor cross-linking. The data shown are the mean ± the standard deviation of triplicate measurements and are representative of five separate experiments. (f) Comparison of the ability of intact anti-FcγRI monoclonal antibodies and F(ab′)2 preparations to induce inositol phosphate generation in dbcAMP-differentiated cells. In the presence of 10 mM LiCl, cells were loaded with either no primary antibody (control), or equivalent concentrations of polyclonal monomeric mouse IgG1 (mlgG1), the F(ab′)2 preparations of the FcγRI-specific monoclonal antibodies 22 and 32 (α-FcγRI F(ab′)2), intact antibodies 22 and 32 (α-FcγRI) or polyclonal monomeric human IgG1 (hlgG1). Following addition of the appropriate secondary antibodies and warming of cells to 37°C for 20 min, total inositol phosphates were measured. The results are the mean ± the standard deviation of triplicate measurements.</note><note type="content">Figure 5: Response of IFN-γ-treated cells to specific cross-linking of FcγRI or FcγRIIa using monoclonal antibodies. (a,b) Accumulation of inositol phosphates at specified times after specific receptor cross-linking. (c,d) DAG generation 30 min after specific receptor cross-linking for the indicated times and the effect of butanol on DAG production. (e) PLD activity after specific receptor cross-linking. The data shown are the mean ± the standard deviation of triplicate measurements and are representative of four separate experiments.</note><note type="content">Figure 6: Sphingosine kinase activity in cells treated with IFN-γ or dbcAMP followed by specific receptor (FcγRI or FcγRIIa) cross-linking using monoclonal antibodies. Controls are IFN-γ-treated cells with no cross-linking antibody added. The data shown are the mean ± the standard deviation of triplicate measurements and are representative of three separate experiments.</note><note type="content">Figure 7: Calcium responses in IFN-γ-and dbcAMP-treated cells following Fcγ receptor aggregation by ligand or cross-linking of specific Fcγ receptors using monoclonal antibodies. (a–f) Changes in Fluo3 fluorescence as an indication of cytosolic calcium – representative traces from three cells (from different experiments) in either (a–c) dbcAMP-differentiated cells or (d–f) IFN-γ-treated cells. (a,d) Fcγ receptor aggregation triggered by cross-linking monomeric human IgG; (b,e) cross-linking of FcγRI using specific monoclonal antibodies 22 and 32; (c,f) cross-linking of FcγRIIa using specific monoclonal antibody 2e1 on cells preloaded with human IgG4 to block the binding site of FcγRI (detailed in Materials and methods). (g) Statistical analysis of the proportion of cells responding with calcium oscillations or a single spike of calcium influx under the conditions shown in (a–f). Each condition – cross-linking using IgG (norm), anti-FcγRI antibodies (FcγRI) or anti-FcγRIIa antibodies (FcγRIIa) – was examined in four separate experiments. The mean value standard error is given for each condition; at least 200 individual cells were analysed.</note><note type="content">Figure 8: Levels of mRNA for FcγRI, FcγRIIa and the γ chain. (a) U937 cells were treated with IFN-γ and cells harvested at 0, 1, 3, 6, 12 and 24 h. Equal amounts of total RNA from each time point were electrophoresed through a formaldehyde 1% agarose gel. After transfer on to nylon membranes, specific transcripts for FcγRI, FcγRIIa and γ chain were visualised using the relevant 32P-labelled probes. (b) U937 cells were treated with dbcAMP and cells harvested at 0, 1, 3, 6, 12 and 48 h. Total RNA extracted from these cells was handled as in (a). The positions of 28S and 18S RNAs are indicated.</note><note type="content">Figure 9: Loading of cells with antisense oligonucleotides demonstrates that FcγRI is coupled to PtdCho-PLD through the recruitment of the γ chain in IFN-γ-treated cells but is coupled to PtdInsP2-PLC through FcγRIIa in dbcAMP-differentiated cells. (a,b) U937 cells were loaded with either antisense γ chain or antisense FcγRIIa prior to treatment with IFN-γ overnight. Specific aggregation of FcγRI or FcγRIIa was achieved using monoclonal antibodies (see Materials and methods for more details). PtdCho-PLD activity (a) and the accumulation of inositol phosphates 20 min after receptor aggregation (b) were measured. The data shown are the mean ± the standard deviation of triplicate measurements. (c) U937 cells were loaded with either antisense γ chain or antisense FcγRIIa prior to treatment for 48 hours with dbcAMP (1 mM). The total accumulation of inositol phosphates was measured in cells 20 min after the specific aggregation of FcγRI or FcγRIIa using monoclonal antibodies. The data shown are the mean ± the standard deviation of triplicate measurements. (d) IFN-γ-primed U937 cells were loaded with a jumbled antisense control oligonucleotide and the effect on FcγRI coupling to PtdCho-PLD was assessed. (e) DbcAMP-differentiated U937 cells were loaded with the jumbled antisense control oligonucleotide and the effect on FcγRIIa coupling to PtdInsP2-PLC was assessed.</note><note type="content">Figure 10: Diagramatic representation of specific receptor aggregation. (a) Conventional aggregation of antibody receptor. The normal ligand, monomeric human IgG (hIgG), occupies FcγRI through the Fc part of the antibody. This is then aggregated by addition of the F(ab) fragment of goat anti-human IgG F(ab) (GαHIgG). (b) Specific cross-linking of FcγRI. The monoclonal antibodies (mAbs) 22 and 32 specifically recognise FcγRI through their F(ab) region. FcγRI is then aggregated by addition of F(ab) goat anti-mouse IgG F(ab) (GαM). (c) Specific cross-linking of FcγRIIa. The monoclonal antibody 2e 1 specifically recognises FcγRIIa through the F(ab) region. However, this mAb can bind to FcγRI at high affinity through its Fc region as it is a murine IgG2a. To prevent this, the high-affinity receptor is occupied by saturating concentrations of human IgG4 (hIgG4; the Fc region of IgG4 is recognised by FcγRI and not FcγRIIa). The mAb bound to FcγRIIa is then aggregated by addition of the F(ab) fragment of goat anti-mouse IgG F(ab).</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">A molecular switch changes the signalling pathway used by the FcγRI antibody receptor to mobilise calcium</title><author xml:id="author-0000"><persName><forename type="first">Alirio</forename><surname>Melendez</surname></persName><affiliation>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</affiliation></author><author xml:id="author-0001"><persName><forename type="first">R.Andres</forename><surname>Floto</surname></persName><affiliation>Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG, UK</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Angus J.</forename><surname>Cameron</surname></persName><affiliation>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</affiliation></author><author xml:id="author-0003"><persName><forename type="first">David J.</forename><surname>Gillooly</surname></persName><affiliation>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</affiliation></author><author xml:id="author-0004"><persName><forename type="first">Margaret M.</forename><surname>Harnett</surname></persName><affiliation>Department of Immunology, University of Glasgow, Glasgow G11 6NT, Scotland, UK</affiliation></author><author xml:id="author-0005"><persName><forename type="first">Janet M.</forename><surname>Allen</surname></persName><email>janet.allen@bio.gla.ac.uk</email><affiliation>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</affiliation><affiliation>Corresponding author.</affiliation></author><idno type="istex">B24ED7113398FAB8F911958DF2F198CFB7B64BD9</idno><idno type="ark">ark:/67375/6H6-TKRZZVTD-T</idno><idno type="DOI">10.1016/S0960-9822(98)70085-5</idno><idno type="PII">S0960-9822(98)70085-5</idno></analytic><monogr><title level="j">Current Biology</title><title level="j" type="abbrev">CURBIO</title><idno type="pISSN">0960-9822</idno><idno type="PII">S0960-9822(00)X0239-4</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1998"></date><biblScope unit="volume">8</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="210">210</biblScope><biblScope unit="page" to="222">222</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1996</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: Background: Leukocytes express Fcγ receptors, which are specific for the constant region of immunoglobulin G. Aggregation of these receptors activates a repertoire of responses that can lead to targeted cell killing by antibody directed cellular cytotoxicity. The nature of the myeloid response to Fcγ receptor aggregation is highly variable and depends on the maturation state of the cell, but little is known about the signalling mechanisms underlying this variability.Results: We show here that differentiation of a monocytic cell line, U937, to a more macrophage phenotype resulted in an absolute and fundamental switch in the nature of the phospholipid signalling pathway recruited following Fcγ receptor aggregation. In cytokine-primed monocytes, aggregation of the high-affinity receptor FcγRI resulted in the activation of phospholipase D and sphingosine kinase, which in turn led to the transient release of stored calcium; these effects were mediated by the γ chain, an FcγRI accessory protein. In contrast, in cells differentiated to a more macrophage type, aggregation of FcγRI resulted in the FcγRIIa-mediated activation of phospholipase C, and the resulting calcium response was prolonged as calcium entry was stimulated.Conclusions: The switch in FcγRI signalling pathways upon monocyte differentiation is mediated by a switch in the accessory molecule recruited by FcγRI, which lacks its own intrinsic signal transduction motif. As many immune receptors have separate polypeptide chains for ligand binding and signal transduction (allowing a similar switch in signalling pathways), the mechanism described here is likely to be widely used.</p></abstract><textClass><keywords scheme="keyword"><list><head>article-category</head><item><term>Research Paper</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1997-12-18">Modified</change><change when="1998">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-TKRZZVTD-T/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr10" NDATA="IMAGE" name="gr10"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="gr6"></istex:entity><istex:entity SYSTEM="gr7" NDATA="IMAGE" name="gr7"></istex:entity><istex:entity SYSTEM="gr8" NDATA="IMAGE" name="gr8"></istex:entity><istex:entity SYSTEM="gr9" NDATA="IMAGE" name="gr9"></istex:entity><istex:entity SYSTEM="mmc1" NDATA="APPLICATION" name="mmc1"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>CURBIO</jid><aid>18</aid><ce:pii>S0960-9822(98)70085-5</ce:pii><ce:doi>10.1016/S0960-9822(98)70085-5</ce:doi><ce:copyright type="full-transfer" year="1996">Elsevier Science Ltd</ce:copyright><ce:doctopics><ce:doctopic><ce:text>Research Paper</ce:text></ce:doctopic></ce:doctopics></item-info><head><ce:dochead><ce:textfn>Research Paper</ce:textfn></ce:dochead><ce:title>A molecular switch changes the signalling pathway used by the FcγRI antibody receptor to mobilise calcium</ce:title><ce:author-group><ce:author biographyid="BIO1"><ce:given-name>Alirio</ce:given-name><ce:surname>Melendez</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>R.Andres</ce:given-name><ce:surname>Floto</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Angus J.</ce:given-name><ce:surname>Cameron</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>David J.</ce:given-name><ce:surname>Gillooly</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Margaret M.</ce:given-name><ce:surname>Harnett</ce:surname><ce:cross-ref refid="AFF3"><ce:sup>3</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Janet M.</ce:given-name><ce:surname>Allen</ce:surname><ce:cross-ref refid="AFF4"><ce:sup>4</ce:sup></ce:cross-ref><ce:cross-ref refid="COR1">*</ce:cross-ref><ce:e-address type="url">janet.allen@bio.gla.ac.uk</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>1</ce:label><ce:textfn>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>2</ce:label><ce:textfn>Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG, UK</ce:textfn></ce:affiliation><ce:affiliation id="AFF3"><ce:label>3</ce:label><ce:textfn>Department of Immunology, University of Glasgow, Glasgow G11 6NT, Scotland, UK</ce:textfn></ce:affiliation><ce:affiliation id="AFF4"><ce:label>4</ce:label><ce:textfn>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</ce:textfn></ce:affiliation><ce:correspondence id="COR1"><ce:label>*</ce:label><ce:text>Corresponding author.</ce:text></ce:correspondence></ce:author-group><ce:date-received day="14" month="11" year="1997"></ce:date-received><ce:date-revised day="18" month="12" year="1997"></ce:date-revised><ce:date-accepted day="12" month="1" year="1998"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para id="simple-para0055"><ce:bold>Background:</ce:bold> Leukocytes express Fc<ce:italic>γ</ce:italic> receptors, which are specific for the constant region of immunoglobulin G. Aggregation of these receptors activates a repertoire of responses that can lead to targeted cell killing by antibody directed cellular cytotoxicity. The nature of the myeloid response to Fc<ce:italic>γ</ce:italic> receptor aggregation is highly variable and depends on the maturation state of the cell, but little is known about the signalling mechanisms underlying this variability.</ce:simple-para><ce:simple-para id="simple-para0060"><ce:bold>Results:</ce:bold> We show here that differentiation of a monocytic cell line, U937, to a more macrophage phenotype resulted in an absolute and fundamental switch in the nature of the phospholipid signalling pathway recruited following Fc<ce:italic>γ</ce:italic> receptor aggregation. In cytokine-primed monocytes, aggregation of the high-affinity receptor Fc<ce:italic>γ</ce:italic>RI resulted in the activation of phospholipase D and sphingosine kinase, which in turn led to the transient release of stored calcium; these effects were mediated by the <ce:italic>γ</ce:italic> chain, an Fc<ce:italic>γ</ce:italic>RI accessory protein. In contrast, in cells differentiated to a more macrophage type, aggregation of Fc<ce:italic>γ</ce:italic>RI resulted in the Fc<ce:italic>γ</ce:italic>RIIa-mediated activation of phospholipase C, and the resulting calcium response was prolonged as calcium entry was stimulated.</ce:simple-para><ce:simple-para id="simple-para0065"><ce:bold>Conclusions:</ce:bold> The switch in Fc<ce:italic>γ</ce:italic>RI signalling pathways upon monocyte differentiation is mediated by a switch in the accessory molecule recruited by Fc<ce:italic>γ</ce:italic>RI, which lacks its own intrinsic signal transduction motif. As many immune receptors have separate polypeptide chains for ligand binding and signal transduction (allowing a similar switch in signalling pathways), the mechanism described here is likely to be widely used.</ce:simple-para></ce:abstract-sec></ce:abstract></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>A molecular switch changes the signalling pathway used by the FcγRI antibody receptor to mobilise calcium</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>A molecular switch changes the signalling pathway used by the FcγRI antibody receptor to mobilise calcium</title></titleInfo><name type="personal"><namePart type="given">Alirio</namePart><namePart type="family">Melendez</namePart><affiliation>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">R.Andres</namePart><namePart type="family">Floto</namePart><affiliation>Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG, UK</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Angus J.</namePart><namePart type="family">Cameron</namePart><affiliation>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">David J.</namePart><namePart type="family">Gillooly</namePart><affiliation>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Margaret M.</namePart><namePart type="family">Harnett</namePart><affiliation>Department of Immunology, University of Glasgow, Glasgow G11 6NT, Scotland, UK</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Janet M.</namePart><namePart type="family">Allen</namePart><affiliation>Department of Medicine & Therapeutics, Division of Biochemistry & Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK</affiliation><affiliation>Corresponding author.</affiliation><affiliation>E-mail: janet.allen@bio.gla.ac.uk</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1998</dateIssued><dateModified encoding="w3cdtf">1997-12-18</dateModified><copyrightDate encoding="w3cdtf">1996</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: Background: Leukocytes express Fcγ receptors, which are specific for the constant region of immunoglobulin G. Aggregation of these receptors activates a repertoire of responses that can lead to targeted cell killing by antibody directed cellular cytotoxicity. The nature of the myeloid response to Fcγ receptor aggregation is highly variable and depends on the maturation state of the cell, but little is known about the signalling mechanisms underlying this variability.Results: We show here that differentiation of a monocytic cell line, U937, to a more macrophage phenotype resulted in an absolute and fundamental switch in the nature of the phospholipid signalling pathway recruited following Fcγ receptor aggregation. In cytokine-primed monocytes, aggregation of the high-affinity receptor FcγRI resulted in the activation of phospholipase D and sphingosine kinase, which in turn led to the transient release of stored calcium; these effects were mediated by the γ chain, an FcγRI accessory protein. In contrast, in cells differentiated to a more macrophage type, aggregation of FcγRI resulted in the FcγRIIa-mediated activation of phospholipase C, and the resulting calcium response was prolonged as calcium entry was stimulated.Conclusions: The switch in FcγRI signalling pathways upon monocyte differentiation is mediated by a switch in the accessory molecule recruited by FcγRI, which lacks its own intrinsic signal transduction motif. As many immune receptors have separate polypeptide chains for ligand binding and signal transduction (allowing a similar switch in signalling pathways), the mechanism described here is likely to be widely used.</abstract><note type="content">Section title: Research Paper</note><note type="content">Figure 1: Differential generation of inositol phosphates following ligand activation of Fcγ receptor in dbc-AMP- and IFN-γ-treated cells. (a) InsP3 levels in dbc-AMP- or IFN-γ-treated cells at set times (30 sec to 20 min) after Fcγ receptor aggregation. In dbcAMP-differentiated cells, InsP3 concentrations were above control (samples with no added cross-linking antibody) values at all time points after Fcγ receptor aggregation, although concentrations appeared to oscillate. In IFN-γ-treated cells, InsP3 concentrations were never higher than control values at any time point. The data shown are the mean ± the standard deviation of triplicate measurements and are representative of four different experiments. (b) Total accumulated inositol phosphates following Fcγ receptor aggregation in dbcAMP- and IFN-γ-treated cells. Cells were treated with 10 mM lithium chloride. Cells were harvested at 5 min intervals after Fcγ receptor aggregation and assayed for inositol phosphate concentration. The data shown are the mean ± the standard deviation of triplicate measurements and are representative of five separate experiments.</note><note type="content">Figure 2: Relative DAG levels after aggregation of Fcγ receptors by ligand in dbcAMP- and IFN-γ-treated cells. The effect of butanol (0.3%) on the levels of DAG before (control) and after (X link) Fcγ receptor aggregation was examined. The data shown are the mean ± the standard deviation of triplicate measurements and are representative of four separate experiments.</note><note type="content">Figure 3: Measurement of PLD activity in dbcAMP-and IFN-γ-treated cells using the transphosphatidylation assay. (a) PLD activity, measured as the accumulation of [3H]PtdBut in cells before (control) and 30 min after (X link) aggregation of Fcγ receptors. (b) PLD activity in dbcAMP- or IFN-γ-treated cells following activation by phorbol ester (PMA). The data shown are the mean ± the standard deviation of triplicate measurements and are representative of six separate experiments.</note><note type="content">Figure 4: Response of dbcAMP-differentiated cells to specific cross-linking of FcγRI or FcγRIIa using monoclonal antibodies. (a,b) Accumulation of inositol phosphates at specified times after specific receptor cross-linking. (c,d) DAG generation 30 min after specific receptor cross-linking for the indicated times and the effect of butanol on DAG production. (e) PLD activity after specific receptor cross-linking. The data shown are the mean ± the standard deviation of triplicate measurements and are representative of five separate experiments. (f) Comparison of the ability of intact anti-FcγRI monoclonal antibodies and F(ab′)2 preparations to induce inositol phosphate generation in dbcAMP-differentiated cells. In the presence of 10 mM LiCl, cells were loaded with either no primary antibody (control), or equivalent concentrations of polyclonal monomeric mouse IgG1 (mlgG1), the F(ab′)2 preparations of the FcγRI-specific monoclonal antibodies 22 and 32 (α-FcγRI F(ab′)2), intact antibodies 22 and 32 (α-FcγRI) or polyclonal monomeric human IgG1 (hlgG1). Following addition of the appropriate secondary antibodies and warming of cells to 37°C for 20 min, total inositol phosphates were measured. The results are the mean ± the standard deviation of triplicate measurements.</note><note type="content">Figure 5: Response of IFN-γ-treated cells to specific cross-linking of FcγRI or FcγRIIa using monoclonal antibodies. (a,b) Accumulation of inositol phosphates at specified times after specific receptor cross-linking. (c,d) DAG generation 30 min after specific receptor cross-linking for the indicated times and the effect of butanol on DAG production. (e) PLD activity after specific receptor cross-linking. The data shown are the mean ± the standard deviation of triplicate measurements and are representative of four separate experiments.</note><note type="content">Figure 6: Sphingosine kinase activity in cells treated with IFN-γ or dbcAMP followed by specific receptor (FcγRI or FcγRIIa) cross-linking using monoclonal antibodies. Controls are IFN-γ-treated cells with no cross-linking antibody added. The data shown are the mean ± the standard deviation of triplicate measurements and are representative of three separate experiments.</note><note type="content">Figure 7: Calcium responses in IFN-γ-and dbcAMP-treated cells following Fcγ receptor aggregation by ligand or cross-linking of specific Fcγ receptors using monoclonal antibodies. (a–f) Changes in Fluo3 fluorescence as an indication of cytosolic calcium – representative traces from three cells (from different experiments) in either (a–c) dbcAMP-differentiated cells or (d–f) IFN-γ-treated cells. (a,d) Fcγ receptor aggregation triggered by cross-linking monomeric human IgG; (b,e) cross-linking of FcγRI using specific monoclonal antibodies 22 and 32; (c,f) cross-linking of FcγRIIa using specific monoclonal antibody 2e1 on cells preloaded with human IgG4 to block the binding site of FcγRI (detailed in Materials and methods). (g) Statistical analysis of the proportion of cells responding with calcium oscillations or a single spike of calcium influx under the conditions shown in (a–f). Each condition – cross-linking using IgG (norm), anti-FcγRI antibodies (FcγRI) or anti-FcγRIIa antibodies (FcγRIIa) – was examined in four separate experiments. The mean value standard error is given for each condition; at least 200 individual cells were analysed.</note><note type="content">Figure 8: Levels of mRNA for FcγRI, FcγRIIa and the γ chain. (a) U937 cells were treated with IFN-γ and cells harvested at 0, 1, 3, 6, 12 and 24 h. Equal amounts of total RNA from each time point were electrophoresed through a formaldehyde 1% agarose gel. After transfer on to nylon membranes, specific transcripts for FcγRI, FcγRIIa and γ chain were visualised using the relevant 32P-labelled probes. (b) U937 cells were treated with dbcAMP and cells harvested at 0, 1, 3, 6, 12 and 48 h. Total RNA extracted from these cells was handled as in (a). The positions of 28S and 18S RNAs are indicated.</note><note type="content">Figure 9: Loading of cells with antisense oligonucleotides demonstrates that FcγRI is coupled to PtdCho-PLD through the recruitment of the γ chain in IFN-γ-treated cells but is coupled to PtdInsP2-PLC through FcγRIIa in dbcAMP-differentiated cells. (a,b) U937 cells were loaded with either antisense γ chain or antisense FcγRIIa prior to treatment with IFN-γ overnight. Specific aggregation of FcγRI or FcγRIIa was achieved using monoclonal antibodies (see Materials and methods for more details). PtdCho-PLD activity (a) and the accumulation of inositol phosphates 20 min after receptor aggregation (b) were measured. The data shown are the mean ± the standard deviation of triplicate measurements. (c) U937 cells were loaded with either antisense γ chain or antisense FcγRIIa prior to treatment for 48 hours with dbcAMP (1 mM). The total accumulation of inositol phosphates was measured in cells 20 min after the specific aggregation of FcγRI or FcγRIIa using monoclonal antibodies. The data shown are the mean ± the standard deviation of triplicate measurements. (d) IFN-γ-primed U937 cells were loaded with a jumbled antisense control oligonucleotide and the effect on FcγRI coupling to PtdCho-PLD was assessed. (e) DbcAMP-differentiated U937 cells were loaded with the jumbled antisense control oligonucleotide and the effect on FcγRIIa coupling to PtdInsP2-PLC was assessed.</note><note type="content">Figure 10: Diagramatic representation of specific receptor aggregation. (a) Conventional aggregation of antibody receptor. The normal ligand, monomeric human IgG (hIgG), occupies FcγRI through the Fc part of the antibody. This is then aggregated by addition of the F(ab) fragment of goat anti-human IgG F(ab) (GαHIgG). (b) Specific cross-linking of FcγRI. The monoclonal antibodies (mAbs) 22 and 32 specifically recognise FcγRI through their F(ab) region. FcγRI is then aggregated by addition of F(ab) goat anti-mouse IgG F(ab) (GαM). (c) Specific cross-linking of FcγRIIa. The monoclonal antibody 2e 1 specifically recognises FcγRIIa through the F(ab) region. However, this mAb can bind to FcγRI at high affinity through its Fc region as it is a murine IgG2a. To prevent this, the high-affinity receptor is occupied by saturating concentrations of human IgG4 (hIgG4; the Fc region of IgG4 is recognised by FcγRI and not FcγRIIa). The mAb bound to FcγRIIa is then aggregated by addition of the F(ab) fragment of goat anti-mouse IgG F(ab).</note><subject><genre>article-category</genre><topic>Research Paper</topic></subject><relatedItem type="host"><titleInfo><title>Current Biology</title></titleInfo><titleInfo type="abbreviated"><title>CURBIO</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1998</dateIssued></originInfo><identifier type="ISSN">0960-9822</identifier><identifier type="PII">S0960-9822(00)X0239-4</identifier><part><date>1998</date><detail type="volume"><number>8</number><caption>vol.</caption></detail><detail type="issue"><number>4</number><caption>no.</caption></detail><extent unit="issue-pages"><start>R107</start><end>R144</end></extent><extent unit="issue-pages"><start>181</start><end>246</end></extent><extent unit="pages"><start>210</start><end>222</end></extent></part></relatedItem><identifier type="istex">B24ED7113398FAB8F911958DF2F198CFB7B64BD9</identifier><identifier type="ark">ark:/67375/6H6-TKRZZVTD-T</identifier><identifier type="DOI">10.1016/S0960-9822(98)70085-5</identifier><identifier type="PII">S0960-9822(98)70085-5</identifier><accessCondition type="use and reproduction" contentType="copyright">©1996 Elsevier Science Ltd</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Elsevier Science Ltd, ©1996</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-TKRZZVTD-T/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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