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Platelet-mediated inhibition of polymorphonuclear neutrophil apoptosis
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-23981Local ID: 3532ISBN: 91-7373-805-0 (print)OAI: oai:DiVA.org:liu-23981DiVA: diva2:244297
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
List of papers
1. Particles binding β2-integrins mediate intracellular production of oxidative metabolites in human neutrophils independently of phagocytosis
Open this publication in new window or tab >>Particles binding β2-integrins mediate intracellular production of oxidative metabolites in human neutrophils independently of phagocytosis
Show others...
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26025 (URN)10.1016/S0167-4889(99)00123-8 (DOI)10478 (Local ID)10478 (Archive number)10478 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
2. Involvement of the ß2-integrin CD18 in apoptosis signal transduction in human neutrophils
Open this publication in new window or tab >>Involvement of the ß2-integrin CD18 in apoptosis signal transduction in human neutrophils
2000 (English)In: Inflammation Research, ISSN 1023-3830, E-ISSN 1420-908X, Vol. 49, no 9, 452-459 p.Article in journal (Refereed) Published
Abstract [en]

Objective and design: To examine the hypothesis that an accelerated rate of neutrophil apoptosis occurs following β2-integrin activation, and further investigate the signal transduction pathways involved.

Material: Human polymorphonuclear neutrophils.

Treatment: Neutrophils were challenged with pansorbins coated with antibodies towards the β2-integrin subunit CD18 in a proportion of 1:100 with or without the inhibitors diphenylene iodonium (10 M), cytochalasin B (5 μg/ml), genistein (10 nM), herbimycin A (10 M) and Z-VAD-FMK (10 μM).

Methods: Measurement of phosphatidylserine exposure and DNA fragmentation in flow cytometry and assessment of H2O2-production through spectrofluorometry. The results were analysed using Mann Whitney U test and Kruskal Wallis.

Results: Pansorbins coated with antibodies to CD18 induce apoptosis in neutrophils (p < 0.01), and activate the production of reactive oxygen species (p < 0.01). Pre-treatment with the inhibitors have no effect on anti-CD18 induced apoptosis.

Conclusion: Anti-CD18 pansorbins induce apoptosis in neutrophils through an alternative pathway not involving reactive oxygen species and independent of tyrosine phosphorylation, cytoskeletal reorganisation and caspases.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26104 (URN)10.1007/s000110050616 (DOI)10563 (Local ID)10563 (Archive number)10563 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
3. Anti-inflammatory effects of a titanium-peroxy gel: role of oxygen metabolites and apoptosis
Open this publication in new window or tab >>Anti-inflammatory effects of a titanium-peroxy gel: role of oxygen metabolites and apoptosis
2004 (English)In: Journal of Biomedical Materials Research, ISSN 0021-9304, E-ISSN 1097-4636, Vol. 68, no 3, 448-457 p.Article in journal (Refereed) Published
Abstract [en]

Polymorphonuclear neutrophils (PMN) are among the first inflammatory cells to arrive at an implant interface, where they encounter with the foreign material and may produce reactive oxygen species (ROS). During the interaction between titanium and ROS, titanium-peroxy (Ti-peroxy) compounds may be formed. We used a Ti-peroxy gel, made from titanium and hydrogen peroxide, to study the effects of Ti-peroxy compounds on PMN. In the absence of serum, the Ti-peroxy gel decreased the oxidative response of PMN to yeast and PMA and reduced PMN apoptosis without inducing necrosis. These effects could not be ascribed to the release of hydrogen peroxide from the Ti-peroxy gel, because a steady-state hydrogen peroxide producing system failed to mimic the effects of the gel. The effects were similarly unaffected when PMN were preincubated with β2-integrin antibodies, questioning the involvement of adhesion molecules. Nevertheless, when a filter was used to separate the Ti-peroxy gel from the cells, the gel effect on PMN life span was abolished, pointing to a contact-dependent mechanism. In the presence of serum, the Ti-peroxy gel had no effect on the PMN oxidative response and life span, but appeared rather inert. In summary, this study demonstrates that the Ti-peroxy gel has potentially anti-inflammatory properties through a combined peroxide and physical contact effect, supporting the notion that interactions between titanium and inflammatory cells are responsible for the good performance of titanium in vivo.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-23785 (URN)10.1002/jbm.a.20078 (DOI)3302 (Local ID)3302 (Archive number)3302 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
4. Platelet-mediated inhibition of polymorphonuclear neutrophil apoptosis principally involves membranes structures: role of sialyl-Lewisχ epitopes and CD18
Open this publication in new window or tab >>Platelet-mediated inhibition of polymorphonuclear neutrophil apoptosis principally involves membranes structures: role of sialyl-Lewisχ epitopes and CD18
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Objective and design: To clarify the long-time effects of platelets on polymorphonuclear neutrophils (PMNs) apoptosis, with particular emphasis on the involvement of cell adhesion molecules (CAMs).

Material: Isolated human platelets and PMNs.

Treatment: PMNs were treated with antibodies towards adhesion molecules CD18 (2 µg/ml), sialyl-Lewisχ (2 µg/ml) or P-selectin glycoprotein ligand 1 (5 µg/ml) and then incubated in the absence or presence of resting, thrombin-activated or inhibited platelets (PMN:platelet ratio of 1:50), platelet membrane (0.7 mg/ml; equivalent to the 1:50 ratio), or supematant from thrombin-activated platelets.

Methods: Measurement of DNA fragmentation using flow cytometry, microscopical evaluation of adhesion and cell death. Light transmission analysis for recording platelet aggregation.

Results: Activated, and to lesser extent, resting platelets prevented spontaneous PMN apoptosis. Comparable effects were detected by using platelet membrane. Platelet-mediated suppression of PMN apoptosis and PMN-platelet adhesion were reversed by pretreatment with antibodies directed towards the adhesive structures sialyl-Lewisχ (p<0.001).

Conclusions: Our results point to a central role of CAMs in plateletinduced inhibition of PMN apoptosis. Furthermore, the results add new evidences for a close association between the hemostatic and the inflammatory systems.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84797 (URN)
Available from: 2012-10-22 Created: 2012-10-22 Last updated: 2012-10-22Bibliographically approved

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