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Pro- and anti-inflammatory regulation of β2 integrin signalling in human neutrophils
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The body is under constant attack from pathogens trying to slip by our immune defence. If the barrier is breached, invading pathogens enter the tissues and cause inflammation. During this process neutrophils, constituting the first line of defence, leave the bloodstream and seek out and kill the invading pathogens. The mechanisms leading to activation of receptors on neutrophils must be closely orchestrated. Pro- and anti-inflammatory substances can influence the outcome of the inflammation process by affecting the involved players. If not well balanced, inflammatory diseases, such as atherosclerosis and rheumatoid arthritis, can be the outcome.

The aim of this thesis was to elucidate the effect of pro- (fMLP, Leukotriene B4, and Interleukin-8) and anti- (lipoxins, aspirin and statins) inflammatory substances on the β2 integrins, mediating adhesion of neutrophils both under “normal” conditions and during coronary artery disease. More specifically, the effect of these substances on the β2 integrins were studied in regard to: i) the activity (i.e. affinity and avidity) of β2 integrins, ii) the signalling capacity of β2 integrins (i.e. detected as release of arachidonic acid, and the production of reactive oxygen species, and iii) the signal transduction mediated by the β2 integrins (i.e. phosphorylation of Pyk2).

The pro-inflammatory substances belong to the family of chemoattractants that induces transmigration and chemotaxis. A hierarchy exists between the different family members; the end-target chemoattractants (e.g. fMLP) being more potent than intermediary chemoattractants (e.g. IL-8 and LTB4). It was found that intermediary chemoattractants regulate β2 integrins by mainly affecting the avidity of β2 integrins. End-target chemoattractants on the other hand, affected the β2 integrins by increasing the avidity and the affinity, as well as their signalling capacity.

The anti-inflammatory substances used in this study were the exogenous aspirin and statins, and the endogenous lipoxins. In the presence of aspirin, stable analogues of lipoxin (i.e. epi-lipoxins) are formed in a trans-cellular process. Lipoxin inhibited the signalling capacity of β2 integrins mediated by intermediary chemoattractants, as well as the signal transduction induced by end-target chemoattractants. Moreover, the signalling capacity of β2 integrins in neutrophils from patients suffering from coronary artery disease (CAD) was impaired. Arachidonic acid, the precursor for both pro- and anti-inflammatory eicosanoid, induced an increase in the β2 integrin activity (both affinity and avidity), but had no effect on the signal transduction.

In conclusion, different “roles” were observed for end-target and intermediary chemoattractants in the regulation of β2 integrins. The inhibitory effects of the anti-inflammatory lipoxins support earlier studies suggesting that these agents function as “stop signals” in inflammation. This is also confirmed by our findings in CAD patients, who have elevated levels of epi-lipoxins due to aspirin treatment. Moreover, Pyk2 was identified as a possible target for the inhibitory effect of anti-inflammatory drugs.

Place, publisher, year, edition, pages
Linköping: Institutionen för biomedicin och kirurgi , 2007. , 75 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 996
Keyword [en]
inflammation, neutrophils, signalling, integrins
National Category
Cell Biology
Identifiers
URN: urn:nbn:se:liu:diva-9661ISBN: 978-91-85715-22-0 (print)OAI: oai:DiVA.org:liu-9661DiVA: diva2:24065
Public defence
2007-05-16, Berzeliussalen, Hälsouniversitetet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2007-09-13 Created: 2007-09-13 Last updated: 2010-01-14
List of papers
1. Differential inside-out activation of β2 integrins by leukotriene B4 and fMLP in human neutrophils
Open this publication in new window or tab >>Differential inside-out activation of β2 integrins by leukotriene B4 and fMLP in human neutrophils
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2004 (English)In: Experimental Cell Research, ISSN 0014-4827, Vol. 300, no 2, 308-319 p.Article in journal (Refereed) Published
Abstract [en]

We have investigated how LTB4, an endogenous chemoattractant encountered early in the inflammatory process, and fMLP, a bacteria-derived chemotactic peptide emanating from the site of infection, mediate inside-out regulation of the β2-integrin. The role of the two chemoattractants on β2-integrin avidity was investigated by measuring their effect on β2-integrin clustering and surface mobility, whereas their effect on β2-integrin affinity was measured by the expression of a high affinity epitope, a ligand-binding domain on β2-integrins, and by integrin binding to s-ICAM. We find that the two chemoattractants modulate the β2-integrin differently. LTB4 induces an increase in integrin clustering and surface mobility, but only a modest increase in integrin affinity. fMLP evokes a large increase in β2-integrin affinity as well as in clustering and mobility. Lipoxin, which acts as a stop signal for the functions mediated by pro-inflammatory agents, was used as a tool for further examining the inside-out mechanisms. While LTB4-induced integrin clustering and mobility were inhibited by lipoxin, only a minor inhibition of fMLP-induced β2-integrin avidity and no inhibition of integrin affinity were detected. The different modes of the inside-out regulation of β2-integrins suggest that distinct mechanisms are involved in the β2-integrin modulation induced by various chemoattractants.

Keyword
β2-Integrins, Cell adhesion, Chemotactic factors, Eicosanoids, Inflammation, Leukotriene B4, Lipoxins, Human Neutrophils, Signal transduction
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14629 (URN)10.1016/j.yexcr.2004.07.015 (DOI)
Available from: 2007-09-13 Created: 2007-09-13 Last updated: 2009-12-14Bibliographically approved
2. LIpoxin A4 inhibits the fMet-Leu-Phe-induced, but not the β2 integrin-induced activation of the non-receptor tyrosine kinase Pyk2 in Human Leukemia 60 cells
Open this publication in new window or tab >>LIpoxin A4 inhibits the fMet-Leu-Phe-induced, but not the β2 integrin-induced activation of the non-receptor tyrosine kinase Pyk2 in Human Leukemia 60 cells
Manuscript (Other academic)
Identifiers
urn:nbn:se:liu:diva-14630 (URN)
Available from: 2007-09-13 Created: 2007-09-13 Last updated: 2010-01-13
3. Inside-out regulated β2-integrin-induced release of arachidonic acid in Human Leukemia 60 cells
Open this publication in new window or tab >>Inside-out regulated β2-integrin-induced release of arachidonic acid in Human Leukemia 60 cells
Manuscript (Other academic)
Identifiers
urn:nbn:se:liu:diva-14631 (URN)
Available from: 2007-09-13 Created: 2007-09-13 Last updated: 2010-01-13
4. Inactivation of Cdc42 is nessecary for depolymerization of phagosomal F-actin and subsequent phagosomal maturation
Open this publication in new window or tab >>Inactivation of Cdc42 is nessecary for depolymerization of phagosomal F-actin and subsequent phagosomal maturation
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2007 (English)In: Journal of Immunology, ISSN 0022-1767, Vol. 178, no 11, 7357-7365 p.Article in journal (Refereed) Published
Abstract [en]

Phagocytosis is a complex process involving the activation of various signaling pathways, such as the Rho GTPases, and the subsequent reorganization of the actin cytoskeleton. In neutrophils, Rac and Cdc42 are activated during phagocytosis but less is known about the involvement of these GTPases during the different stages of the phagocytic process. The aim of this study was to elucidate the role of Cdc42 in phagocytosis and the subsequent phagosomal maturation. Using a TAT-based protein transduction technique, we introduced dominant negative and constitutively active forms of Cdc42 into neutrophil-like HL60 (human leukemia) cells that were allowed to phagocytose IgG-opsonized yeast particles. Staining of cellular F-actin in cells transduced with constitutively active Cdc42 revealed that the activation of Cdc42 induced sustained accumulation of periphagosomal actin. Moreover, the fusion of azurophilic granules with the phagosomal membrane was prevented by the accumulated F-actin. In contrast, introducing dominant negative Cdc42 impaired the translocation per se of azurophilic granules to the periphagosomal area. These results show that efficient phagosomal maturation and the subsequent eradication of ingested microbes in human neutrophils is dependent on a strictly regulated Cdc42. To induce granule translocation, Cdc42 must be in its active state but has to be inactivated to allow depolymerization of the F-actin cage around the phagosome, a process essential for phagolysosome formation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14632 (URN)
Available from: 2007-09-13 Created: 2007-09-13
5. Neutrophil activation status in stable coronary artery disease.
Open this publication in new window or tab >>Neutrophil activation status in stable coronary artery disease.
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2007 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 2, no 10, e1056- p.Article in journal (Refereed) Published
Abstract [en]

Background: During the last years, neutrophils have emerged as important players in atherogenesis. They are highly activated in peripheral blood of patients with unstable angina. Moreover, a primed state of circulating neutrophils has been proposed in patients with stable angina. Our aim was to investigate the neutrophil activation status in patients with stable coronary artery disease (CAD) at conventional drug treatment.

Methodology and principal findings: Thirty patients with stable CAD and 30 healthy controls were included using a paired design. The neutrophil expression of CD18 and high-affinity state of CD11b was analysed by flow cytometry before and after stimulation with chemoattractants. Also, the production of reactive oxygen species (ROS) was determined by chemiluminescence. During basal conditions, the neutrophil expression of CD18 or high-affinity state of CD11b did not differ between patients and controls. Chemoattractants (Interleukin-8 and Leukotriene B(4)) did not increase either the expression or the amount of high-affinity CD11b/CD18-integrins in CAD patients compared to controls, and had no effect on the production of ROS. On the other hand, the ROS production in response to C3bi-opsonised yeast particles and the neutrophils' inherent capacity to produce ROS were both significantly decreased in patients.

Conclusion/Significance: We could not find any evidence that neutrophils in patients with stable CAD were primed, i.e. more prone to activation, compared to cells from healthy controls. According to our data, the circulating neutrophils in CAD patients rather showed an impaired activation status. It remains to be elucidated whether the neutrophil dysfunction in CAD is mainly a marker of chronic disease, an atherogenic factor or a consequence of the drug treatment.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-17246 (URN)10.1371/journal.pone.0001056 (DOI)17957240 (PubMedID)
Available from: 2009-03-12 Created: 2009-03-12 Last updated: 2010-01-14

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