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Radical aspects on arthritis: the role of neutrophil generation of nitric oxide and superoxide in inflammatory conditions
Linköping University, Department of Clinical and Experimental Medicine, Rheumatology . Linköping University, Faculty of Health Sciences.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The polymorphonuclear neutrophil granulocytes (neutrophils) are gaining renewed interest regarding their involvement in chronic inflammatory disorders, including rheumatoid arthritis (RA). Besides phagocytic and destructive capabilities, neutrophils have regulatory roles, e.g. by influencing responses from dendritic cells and lymphocytes. Several animal models have revealed that neutrophils are crucial for the initiation and maintenance of chronic inflammatory diseases. Neutrophil function is highly dependent on their ability to produce superoxide, an oxygen radical which can be further metabolized to other free radicals. Whether or not neutrophils are capable of producing the oxygen radical nitric oxide (NO˙) has been a matter of debate.

In this thesis it was shown that freshly isolated neutrophils from the joint cavity of patients with RA, but not from other arthritis patients, had ongoing intracellular production of superoxide, indicating the processing of ingested material.

The finding that joint neutrophils, but seemingly not circulating cells, expressed the NO-inducing enzyme iNOS, led to a series of experiments aimed to elucidate where in the exudative process this enzyme could first be detected. We could finally, for the first time, present evidence that human neutrophils actually express iNOS constitutively. Our data also suggest that neutrophil iNOS may be membrane associated, thus differing from the cytosolic location in other cell types. Since NOS activity was not demonstrated in isolated cells, the notion that neutrophil iNOS is regulated primarily at the transcriptional level must be questioned. NO production from iNOS requires the presence of its substrate, L-arginine. To test the hypothesis that neutrophil arginase prevents neutrophil NO-production, we investigated whether arginase inhibition affects neutrophil NO-dependent oxidative function. Initial data revealed a difference in the effect of arginase inhibition comparing neutrophil stimulus with a soluble formylated tri-peptide (fMLF) and integrin-mediated stimulation with particle-bound collagen type-1. This led to the hypothesis that integrin-ligation on neutrophils induces extracellular liberation of arginase, which was confirmed both by measuring arginase and its enzyme activity. The findings in this thesis may be important not only regarding the role of neutrophils in chronic joint inflammation, but also as a link in the accelerated atherosclerosis observed in chronic inflammatory disorders, e.g. RA.

Abstract [sv]

Vid reumatiska ledinflammationer ansamlas mycket stora mängder polymorfkärniga neutrofila granulocyter (neutrofiler) inne i den vätskefyllda ledhålan. Neutrofiler har kraftfull destruktiv potential och anses kunna bidra till uppkomst av skada i leden. Då flera djurmodeller av ledinflammation har visat sig omöjliga att initiera i frånvaro av neutrofiler, har intresset för denna celltyp åter ökat efter att de under lång tid har stått i skuggan av andra typer av vita blodkroppar. En viktig del i avdödning av mikroorganismer och cellsignalering är förmågan att bilda fria syreradikaler, t.ex. superoxid (˙O2-) och kväveoxid (NO˙). Denna avhandling belyser aspekter kring produktionen av dessa reaktiva syreprodukter och mekanismer av potentiell betydelse vid ledinflammation.

I det första arbetet visas att neutrofiler isolerade ur ledvätska från patienter med ledgångsreumatism (RA) har ett unikt beteende avseende superoxidproduktion jämfört med motsvarande celler från patienter med andra reumatiska sjukdomar. RA-neutrofiler från ledvätska (men inte från blod) producerar superoxid intracellulärt redan i vila och stimulering via vidhäftningsmolekyler ger en snabb ytterligare ökning av denna aktivitet. Fyndet antyder att cellerna är engagerade med hantering av endocyterade partiklar och/eller immunkomplex/immunaggregat.

I de båda nästkommande arbetena undersöktes förekomst av det NO˙-producerande enzymet iNOS i neutrofiler. En rad tidigare publikationer har rapporterat motsägelsefulla resultat i denna fråga. Efter en serie experiment kunde vi konstatera att humana neutrofiler uttrycker iNOS konstitutivt, men att både dess cellulära lokalisation och reglering skiljer sig från andra celler.

Neutrofiler har nyligen även visats innehålla arginas, ett enzym som konkurrerar med iNOS om bindningen till L-arginin och som därmed kan hämma NO˙-produktion. I det fjärde arbetet undersökte vi därför om hämning av arginas påverkade neutrofilernas funktion och produktion av superoxid. Vi fann att effekterna av arginashämning var större hos celler som stimulerats genom vidhäftning av kollagenklädda partiklar jämfört med en löslig formylerad tri-peptid (fMLF). Vidare, kunde vi visa att vidhäftning av kollagenklädda partiklar medför större extracellulär frisättning av arginas. Med stöd av dessa fynd kunde vi i påföljande försök bekräfta hypotesen att extracellulär frisättning av arginas är större efter vidhäftning av kollagen-partiklar än med fMLF-stimulering. Fysiologiskt är fyndet logiskt då det skulle medföra ökade vidhäftningsmöjligheter för neutrofilen inne i blodbanan genom att begränsa blodkärlets egen NO˙ produktion. Fyndet är också förenligt med den ökade frekvensen hjärt- och kärlsjukdomar vid RA, då en intensiv kontinuerlig utvandring av neutrofiler skulle medföra ökad arginas frisättning, sänkta argininnivåer och endotelial dysfunktion.

Place, publisher, year, edition, pages
Institutionen för molekylär och klinisk medicin , 2007.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 984
Keyword [en]
neutrophils, reactive oxygen species, arthritis, NOS, NO, arginase, integrins
National Category
Clinical Medicine
Identifiers
URN: urn:nbn:se:liu:diva-9505ISBN: 978-91-85715-67-1 (print)OAI: oai:DiVA.org:liu-9505DiVA: diva2:23959
Public defence
2007-03-16, Berzeliussalen, Hälsouniversitetet, Universitetssjukhuset, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2007-08-27 Created: 2007-08-27 Last updated: 2015-08-31
List of papers
1. Intracellular oxidative activation in synovial fluid neutrophils from patients with rheumatoid arthritis but not from other arthritis patients
Open this publication in new window or tab >>Intracellular oxidative activation in synovial fluid neutrophils from patients with rheumatoid arthritis but not from other arthritis patients
2007 (English)In: Journal of Rheumatology, ISSN 0315-162X, E-ISSN 1499-2752, Vol. 34, no 11, 2162-2170 p.Article in journal (Refereed) Published
Abstract [en]

Objective: To compare total and intracellular oxidative activation of blood and synovial fluid (SF) neutrophils from patients with rheumatoid arthritis (RA) and other arthritides with blood donor neutrophils.

Methods: Peripheral blood and SF samples were obtained from 26 gonarthritis patients (13 RA, 13 non-RA) attending the rheumatology unit for therapeutic joint aspiration. Isolated neutrophils were stimulated by a formylated tripeptide (fMLF) or by microbeads coated with collagen-I. Formation of superoxide-anion-derived reactive oxygen species (ROS) was studied by luminol-enhanced chemiluminescence. Paired samples of blood and SF neutrophils from patients with active arthritis were compared with blood neutrophils from patients in remission and from 47 healthy blood donors.

Results: SF neutrophils from patients with RA, but not from non-RA patients, showed high baseline intracellular ROS production. Blood neutrophils from arthritis patients in remission existed in a primed state as revealed by more rapid oxidative response after collagen-bead challenge and a more pronounced response after fMLF stimulation compared to healthy blood donors. Blood neutrophils from RA patients with ongoing gonarthritis, however, did not differ from healthy blood donors concerning oxidative activation, whereas blood neutrophils from non-RA patients with gonarthritis showed a significantly lower peak ROS production.

Conclusions: A novel finding with pathogenetic implications in our study is that SF neutrophils from patients with RA, but not other arthritides, are activated and produce ROS intracellularly. This implies that synovial neutrophils in RA are engaged in the processing of endocytosed material.

Keyword
Neutrophils, Arthritis, Reactive oxygen species, Superoxide anion
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14585 (URN)
Available from: 2007-08-27 Created: 2007-08-27 Last updated: 2017-12-13
2. Inducible nitric oxide synthase is expressed in synovial fluid granulocytes
Open this publication in new window or tab >>Inducible nitric oxide synthase is expressed in synovial fluid granulocytes
2002 (English)In: Clinical Experimental Immunology, ISSN 0009-9104, Vol. 130, 150-155 p.Article in journal (Refereed) Published
Abstract [en]

The objective of the study was to evaluate the NO-producing potential of synovial fluid (SF) cells. SF from 15 patients with arthritis was compared with blood from the same individuals and with blood from 10 healthy controls. Cellular expression of inducible nitric oxide synthase (iNOS) was analysed by flow cytometry. High-performance liquid chromatography was used to measure L-arginine and L-citrulline. Nitrite and nitrate were measured colourimetrically utilizing the Griess' reaction. Compared to whole blood granulocytes in patients with chronic arthritis, a prominent iNOS expression was observed in SF granulocytes (P < 0.001). A slight, but statistically significant, increase in iNOS expression was also recorded in lymphocytes and monocytes from SF. L-arginine was elevated in SF compared to serum (257 ± 78 versus 176 ± 65 µmol/l, P = 0.008), whereas a slight increase in L-citrulline (33 ± 11 versus 26 ± 9 µmol/l), did not reach statistical significance. Great variations but no significant differences were observed comparing serum and SF levels of nitrite and nitrate, respectively, although the sum of nitrite and nitrate tended to be elevated in SF (19.2 ± 20.7 versus 8,6 ± 6.5 µmol/l,P = 0.054). Synovial fluid leucocytes, in particular granulocytes, express iNOS and may thus contribute to intra-articular NO production in arthritis.

Keyword
arthritis, granulocytes, iNOS, nitric oxide
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14586 (URN)10.1046/j.1365-2249.2002.01959.x (DOI)
Available from: 2007-08-27 Created: 2007-08-27 Last updated: 2015-08-31
3. Inducible nitric oxide synthase (NOS II) is constitutive in human neutrophils
Open this publication in new window or tab >>Inducible nitric oxide synthase (NOS II) is constitutive in human neutrophils
Show others...
2003 (English)In: APMIS, ISSN 0903-4641, Vol. 111, no 10, 963-968 p.Article in journal (Refereed) Published
Abstract [en]

The objective was to study the expression of inducible nitric oxide synthase (NOS II) in and NO production by human blood neutrophils and in in vivo exudated neutrophils. Cellular expression of NOS II was evaluated by flow cytometry in whole blood, in isolated blood neutrophils, and in neutrophils obtained by exudation in vivo into skin chambers. Neutrophil NOS II was also demonstrated by Western blotting. Uptake of 3H-labelled L-arginine was studied in vitro and NOS activity measured in a whole cell assay by the conversion of 3H-arginine to 3H-citrulline. In contrast to unseparated blood cells, NOS II was demonstrable both in isolated blood neutrophils and exudated cells. The failure to detect NOS II by flow cytometry in whole blood cells thus proved to be due to the quenching effect of hemoglobin. Western blotting revealed a 130 kD band corresponding to NOS II in isolated blood neutrophils, but detection was dependent on diisopropylfluorophosphate for proteinase inhibition. L-arginine was taken up by neutrophils, but enzymatic activity could not be demonstrated. We conclude that human neutrophils constitutively express NOS II, but that its demonstration by FITC-labelling is inhibited by hemoglobin-mediated quenching in whole blood samples.

Keyword
Inflammation, nitric oxide, iNOS, granulocytes
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14587 (URN)10.1034/j.1600-0463.2003.1111008.x (DOI)
Available from: 2007-08-27 Created: 2007-08-27 Last updated: 2015-08-31
4. Oxidative activation of human neutrophils by type-1-collagen-coated particles is influenced by nitric oxide production and modulated by endogenous arginase
Open this publication in new window or tab >>Oxidative activation of human neutrophils by type-1-collagen-coated particles is influenced by nitric oxide production and modulated by endogenous arginase
2007 (English)In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673Article in journal (Refereed) Submitted
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14588 (URN)
Available from: 2007-08-27 Created: 2007-08-27 Last updated: 2017-12-13

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