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Particles binding β2-integrins mediate intracellular production of oxidative metabolites in human neutrophils independently of phagocytosis
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
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1999 (English)In: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, E-ISSN 1879-2596, Vol. 1452, no 2, 133-144 p.Article in journal (Refereed) Published
Abstract [en]

Complement-opsonised particles are readily ingested by human neutrophils through a complement receptor-mediated process leading to phagolysosome fusion and production of oxidative metabolites. To investigate the complement receptor 3 (CR3)-associated signal system involved, cells were challenged with protein A-positive, heat-killed Staphylococcus aureus to which antibodies with specificity for the subunits of the β2-integrins, i.e. anti-CD11b (the α subunit of CR3) and anti-CD18 (the β subunit of CR3), were bound through their Fc moiety. Despite not being ingested by the neutrophils, the surface associated anti-CD18- and anti-CD11b-coated particles were able to activate the neutrophil NADPH-oxidase. Also anti-CD11a- (the α subunit of LFA-1) and to a lesser extent anti-CD11c- (the α subunit of CR4) coated particles were able to trigger the NADPH-oxidase. The NADPH-oxidase was activated without extracellular release of reactive oxygen species. The activity was inhibited by cytochalasin B, suggesting a necessary role for the cytoskeleton in the signalling pathway that activates the oxidase. We show that particle-mediated cross-linking of β2-integrins on the neutrophil surface initiates a signalling cascade, involving cytoskeletal rearrangements, leading to an activation of the NADPH-oxidase without phagosome formation or extracellular release of reactive oxygen species.

Place, publisher, year, edition, pages
1999. Vol. 1452, no 2, 133-144 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26025DOI: 10.1016/S0167-4889(99)00123-8Local ID: 10478OAI: oai:DiVA.org:liu-26025DiVA: diva2:246573
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Regulation of phagocytosis and phagolysosome fusion in human leukocytes
Open this publication in new window or tab >>Regulation of phagocytosis and phagolysosome fusion in human leukocytes
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Professional phagocytes such as neutrophil granulocytes and macrophages are an esential part of the innate immune system. The neutrophils form the first line of defence against invading microorganisms and are important for rapid killing of the intruders. Macrophages arrive later at the site of infection, kill micoorganisms, degrade dead cells, present antigens and secrete substances that orchestrate the inflammatory response. Neutrophils and macrophages ingest and kill microorganisms in a process called phagocytosis, where calcium signalling has shown to be involved. Inside the cell the microorganism is enclosed in a phagosome, that sequentially fuses with various intracellular vesicles to form a phagolysosome in which the intruder is killed. Killing is achieved through the actions of lytic enzymes, nitrogen oxide (NO) and reactive oxygen metabolites (ROM). Studies on the regulation of phagocytosis are essential since many pathogens are able to survive by interfering with this process. In the first study we investigated intracellular signalling in human neutrophils following engagement of a phagocytic receptor, complement receptor 3 (CR3). For this, we used antibody-coated PANSORBINS® which bound to the ß-chain of CR3 without inducing phagocytosis. We found that these particles elicited an intracellular production of ROM which was dependent on the cytoskeleton and on phospholipase D. In the second study, we showed that the putative calcium-sensor synaptotagmin II is present in neutrophils and is involved in phagocytosis. Synaptotagmin II was found on the specific granules and translocated to the phagosome in a calcium-dependent manner during eR-mediated phagocytosis and to the plasma membrane after stimulation with the formylated peptide, N-formyl-methionyl-leucyl-phenylalanine. In the third study, we demonstrate the presence of synaptotagmin IV in human macrophages. Synaptotagmin IV translocated transiendy to macrophage phagosomes during eR- and FcγR-mediated phagocytosis. We also found that eR- and FcyR-mediated uptake was calcium dependent in these cells. In the fourth study, we show that lipophosphoglycan (LPG) from Leishmania donovani induced elevated levels of periphagosomal F-actin, inhibition of phagolysosome maturation and diminished production of ROM in neutrophils during eR-mediated phagocytosis. Together, our data show that generation of ROM occurs early during eR-mediated phagocytosis and could be involved in intracellular signalling, that synaptotagmins are present in professional phagocytes and could act as calcium sensors in phagosomal maturation and secretion, and that LPG can be used as a tool to investigate how actin can regulate phagosomal maturation in neutrophils.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2003. 64 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 818
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26661 (URN)11227 (Local ID)91-7373-509-4 (ISBN)11227 (Archive number)11227 (OAI)
Public defence
2003-11-06, Aulan, Hälsans Hus, Universitetet, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-17Bibliographically approved
2. Platelet-mediated inhibition of polymorphonuclear neutrophil apoptosis
Open this publication in new window or tab >>Platelet-mediated inhibition of polymorphonuclear neutrophil apoptosis
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Neutrophils play an important role in the cell-mediated immune response against invading micro-organisms. Upon infection, neutrophils in the bloodstream transmigrate into the tissue where they bind to and engulf the microbes. The infecting agent is then eliminated using reactive oxygen species (ROS) and proteolytic enzymes. It is crucial that the life span of activated neutrophils is tightly regulated since the prolonged release of ROS can cause severe damage to the surrounding tissue. Therefore, programmed cell suicide through apoptosis, and the subsequent ingestion of apoptotic neutrophils by neighbouring cells, are essential to the rapid resolution of inflammation.

For the apoptotic process to work according to plan, an extensive control system is at work, instructing the neutrophil when and where to die. This system is influenced by intracellular as well as extracellular signals, that are conveyed through the neutrophil plasma membrane by cell adhesion molecules, for example integrins. The aim of this thesis was to investigate the involvement of adhesion molecules in the regulation of the neutrophil ROS production and apoptosis. We found that when neutrophils were challenged with particles exposing antibodies towards ß2-integrins they produced intracellular ROS, and this was dependent on reorganisation of the cytoskeleton. The particles also induced caspase-independent neutrophil apoptosis, and this ß2-integrin mediated signalling seemed to occur independently of tyrosine phosphorylation.

Interaction of neutrophils with a potential biomaterial, a titanium-peroxy gel, reversibly inhibited ROS production and decreased spontaneous apoptosis. This effect was contact-dependent but involved cell signalling via adhesion molecules other than ß2-integrins. We also showed that platelet-induced inhibition of neutrophil apoptosis involved both ß2-integrins and carbohydrate structures, and that activated platelets were more efficient than resting platelets. Platelet membranes also inhibited neutrophil apoptosis, supporting the notion of adhesion molecule involvement.

In conclusion, this thesis shows that adhesion molecules are involved in the regulation of neutrophil ROS production and apoptosis. Adhesion molecule signalling pathways may therefore be potential new therapeutic targets for treatment of diseases involving acute inflammation.

Abstract [sv]

Neutrafiler spelar en viktig roii i kroppens cellmedierade immunförsvar mot invaderande mikroorganismer. Vid en infektion vandrar neutrafilerna från blodet ut i vävnaden där de binder till och äter upp mikroberna. Dessa elimineras sedan med hjälp av reaktiva syremetaboliter och nedbrytande enzymer. Det är viktigt att livslängden för aktiverade neutrofiler kontrolleras noga eftersom långvarig frisättning av reaktiva syremetaboliter kan orsaka allvarliga skador på den omgivande vävnaden. Därför är programmerat självmord genom apoptos, och det faktum att apoptotiska neutrafiler äts upp av kringliggande celler, så viktigt för att en inflammation ska läka ut.

För att apoptosprocessen ska fungera som det är tänkt krävs ett omfattande kontrollsystem som talar om för neutrafilen när och var den ska dö. Systemet påverkas av både intra- och extracellulära signaler som överförs geno:ni neutrafilens plasamamembran av adhesionsmolekyler, tex integriner. Syftet med den här avhandlingen var att undersöka betydelsen av adhesionmolekyler i regleringen av neutrafilers apoptos och produktion av reaktiva syremetaboliter. Vi fann att när neutrafiler interagerade med partiklar som exponerar antikroppar mot ß2-integriner så producerade de reaktiva syremetaboliter intracellulärt. Denna signalväg krävde omlagringar i cytoskelettet. Partiklarna inducerade också caspasoberoende apoptos i neutrafilerna och denna ß2-integrin medierade signalering tycktes ske oberoende av tyrosinfosforylering.

Interaktion mellan neutrafiler och ett potentiellt biomaterial, en titanperoxidgel, gav en reversibel hämning av produktionen av reaktiva syrernetaboliter och minskad spontanapoptas. Effekten var kontaktberoende men signalerna gick via andra adhesionsmolekyler än ß2-integriner. Vi såg också att trombocytmedieract hämning av neutrofilapoptos involverade både ß2-integriner och kolhydratstrukturer, och att aktiverade trombocyter var effektivare än vilande trombocyter. Membran från trombocyter hämmade också neutrofilapoptos vilket stöder iden om att adhesionsmolekyler är inblandade.

Sammanfattningsvis visar den här avhandlingen att adhesions-molekyler är inblandade i regleringen av neutrafilers apoptos och produktion av reaktiva syremetaboliter. De signalvägar som används av adhesionsmolekylerna skulle därmed kunna utgöra nya mål för mediciner som används vid sjukdomar där akut inflammation är inblandad.

Place, publisher, year, edition, pages
Linköping: Linköping Universitet, 2004. 70 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 833
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-23981 (URN)3532 (Local ID)91-7373-805-0 (ISBN)3532 (Archive number)3532 (OAI)
Public defence
2004-02-26, Victoriasalen, Universitetssjukhuset, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-22Bibliographically approved

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Serrander, LenaLarsson, JennyLundqvist, HelenLindmark, MariaStendahl, Olle

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