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Leukocyte sequestration associated with inflammation: mechanisms and modulations
Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [sv]

Inflammationsreaktionen är kroppens första försvar mot främmande partiklar t ex bakterier. Det är en komplex reaktion som bl a karakteriseras av att vita blodkroppar dras till det inflammerade området och att vätskao i blodet lättare tar sig igenom kärlväggen till vävnaden dvs det svullnar. Neutrafiler är en vit blodkropp, som samlas vid inflammationer och oskadliggör fi·ämmaode partiklar genom att fagocytera ("äta upp") dem, släppa ut giftiga ämnen och bilda fria syreradikaler. För att komma från blodet till den inflaonnerade vävnaden fastnar neutrofilema på kapillärväggen och tar sig sen igenom väggen. Vid inflammatoriska sjukdomar t ex reumatism ansamlas också neutrofiler, men angriper då istället kroppens egen vävnad. Vissa kliniska behaodlingar orsakar också att neutrafiler ansamlas t ex i lungorna vid hemodialys, men det är oklart om de har någon skadaode effekt på lungvävnaden. Det är däremot känt att en del hemodialyspatienter efter flera års behaodliog har problem med lungorna. Att förstå och blockera mekanismerna för ansamling av neutrafiler har därför stor betydelse vid många sjukdomar.

I delarbete l studerades vilken effekt ansamlade neutrafiler har på lungvävnaden och lungfunktionen och för detta ändarnål utvecklades en variant av en försöksuppställning med en marsvinslunga upphängd i ett glaskärl. Lungan "andades", cirkulerades med blod och flera parametrar registrerades hela tiden. Modellen visade sig vara stabil med lungor som bl a var mindre vätskefyllda än i tidigare studier och hade ett högt syreinnehåll trots "andning" med rumsluft. För att påvisa systemets känslighet, tillfördes två substanser med känd effekt på kärlväggarna, histamin och oljesyra. Lungorna som fick oljesyra eller histamin reagerade som väntat med ökat kärhnotstånd, ökad genomsläpplighet för vätska genom kärlväggen samt ökat neutroftlinnehåll i lungan, vilket bevisade att lungmodellen var en pålitlig och känslig metod.

I delarbete 2 simulerades en dialysbehaodliog med dialysmembraoet Cuprophan i lungmodellen, för att studera vad som händer i lungan när neutrafiler ansamlas. Hos hemodlalys-patienter fastnar flertalet av alla cirkulerande neutrafiler ca 15 min efter dialysstart och lossnar sedan efterhand under behandlingen. I lungmodellen visade det sig dock att utao dialysmembran fastnade ca 70 % av det totala antalet cirkulerande neutrafiler i lungan och var kvar under de två timmar långa försöken. För att kunna studera om lungvävnaden och andningen förändrades när neutrafilerna fastnade i lungao, järnfördes därför lungor med cirkolation av blod utao vita blodkroppar med lungor med cirkulation av normalt blod. Resultaten tydde på att neutrafiler kao fastna vid kapillärväggama i lungao utan att påverka genomsläppligheten för vätska eller bildaodet av fiia syreradikaler. Neutrafilerna fastnade aotagligen inte via de molekyler som nmmalt langar upp cirkulerande neutrafiler vid inflammationer. Dialysmembranet lyckades inte heller mätbart aktivera neutrofilema, men trots det har en del hernodialyspatienter som fått dialys länge vätskeansamling i lungorna.

Förflyttningen av neutrafiler mot en inflammation startar bl a av att ämnena TNF-α och IL-1ß utsöndrade från inflammationsområdet stimulerar kapillärväggens celler, endotelcellema. I endatelcellerna aktiveras då faktorn NF-κB och celleroa börjar producera olika änmen för att locka till sig cirkulerande neutrafiler bl a släpper de ut ämnet IL-8 och molekylerna selekliner bildas på deras yta. IL-8 är specialiserad på att dra till sig neutrofiler och t ex patienter med inflammatoriska sjukdomar och autoimmuna sjukdomar typ reumatism har forhöjda halter av IL-8. De cirkulerande neutroftlerna hromsas av molekylerna selekliner på endotelcellemas yta och binder löst till dem via kolhydratstrukturer på sin egen yta. Denna bindning leder i sin tur till att IL-8 kan binda till sin receptor på neutrofilerna. Nu ökar molekylerna ß2-integriner på neutrofilemas yta och dessa binder hårt till en annan molekyl på endotelcellema.

I delarbete 3 undersöktes om ämnena interferoner och glukokortikosteroider kunde hämma IL-8 utsläppet från endotelceller. Glukokortikosteroider är mycket använda som läkemedel mot inflammationer och deras anti-inflammatoriska effekt fungerar antagligen via NF-κB. Interferon er är ett ämne i kroppen, som har en invecklad roll vid inflammationer men som också används vid behandling av flera sjukdomar. Humana endotelceller frän navelsträngsvener (HUVEC) stimulerades med IL-1ß eller TNF-α för att simulera en inflammationsreaktion. Både stimulering med IL-1ß och TNF-α resulterade i en enorm ökning av utsläppt IL-8. Vid stimuleringen tillsattes också tre olika interferoner och två glukokortikosteroider. Alla interferoner och steroider hännnade utsläppet av IL-8 i forhållande till dosen. Med immunoflourescence fotograferades också hur IL-8 molekylerna var placerade inuti cellen. Resultatet tydde på att det mesta av bildat IL-8 släpps ut direkt och ej lagras i cellen.

I delarbete 4 undersöktes om interferoner och glukokortikosteroider också hännnade bildandet av IL-8 genom att mäta mängden IL-8 inuti HUVEC med teknikerna ELISA och flödescytometri. För att kunna mäta den totala bildningen av IL-8 blockerades utsläppet med ämnet monensin. Förutom mätning av färdiga IL-8 molekyler undersöktes också med metoden RT-PCR om genen för IL-8 aktiverats. Både interferoner och glukokortikosteroider visade sig vara effektiva hämmare av IL-8 produktionen som var igångsatt av TNF-α. Interferon hämmade dessutom den lilla mängd IL-8 som bildades i ostimulerade endotelceller, vilket antydde att det finns flera sätt att stimulera produktionen än via NF-κB.

Sammanfattningsvis kan sägas att detta arbete visat att vita blodkroppar under vissa forhållande kan ansamlas vid kapillärväggen i lungan, utan att vara så mycket aktiverade att de påverkar kärlväggen. Om neutrafilerna däremot är aktiverade och fastnar via inblandning av IL-8 sknlle glukokortikosteroider och interferoner eventuellt kunna vara möjliga att använda som medicin för att motverka oönskade inflammationsreaktioner.

Abstract [en]

The inflammatory response is the first line of defence against injurious agents. It is a multi-faceted response involving several mechanisms orchestrated by inflammatory mediators such as cytokines, chemokines and adhesion molecules. The inflammatory response is characterised by leukocytes migrating through the endothelium towards the site of inflammation as well as increased capillary permeability resulting in oedema. ln some diseases such as autoimmune diseases and chronic inflammation the inflammatory response is inappropriately triggered and may be harmful to the tissue. In addition, some clinical procedures e.g. haemodialysis cause leukocytes to sequester in the lung, which may affect the pulmonary microvasculature. In this thesis, the effects and mechanisms of leukocyte sequestration were studied ex vivo and, on a molecular level, in vitro in order to get a broader understanding of the inflarmnatory response. The results indicated that leukocytes were able to sequester in the lung microvasculature without affecting the capillary permeability, microvascular resistance or release of oxygen free radicals in the isolated blood-perfused guinea-pig lung. It seemed like the neutrophils were sequestered due to mechanical forces rather than activation, since the model turned out to be a sensitive method for measuring microvascular and respiratory variables in a wellcontrolled environment with all cellular interactions maintained in the tissue. Haemodialysis treatment was simulated in this model, but the connection of a dialysis membrane in the perfusion system did not seem to activate neutrophils to the extent that they generate oxygen free radicals or alter capillary permeability. Nevertheless inappropiate sequestration of neutrophils can be hannful in several diseases and therapeutic modulation of leukocyte sequestration can therefore be of interest IL-8 is a chemokine involved in the inflammatory response, mainly attracting neutrophils to the site of intlannnation. Different cells including endothelial cells produce IL-8. In this study we stimulated endothelial cells with the inflannnatory cytokines IL-1ß or TNF-α to simulate the inflammatory response. It resulted in an increased expression of adhesion molecules as well as dramatically increased secretion and production of IL-8. The present study showed that cytokine-induced IL-8 secretion and production could be inhibited by the innnunomodulatory cytokine IFN-γ and anti-intlannnatory glucocorticosteroids. Steroids and intetferons are used therapeutically in the treatment of autoimmune and inflammatory disorders, although the exact mechanisms of inhibition are not fully elucidated. Taken together, results from ELISA, flow cytornetry and RT-PCR suggested that IFN-γ and corticosteroids inhibit synthesis rather than intracellular transport of IL-8. Their inhibitory effect is probably mediated by the transcription factor NF-K'B, since this factor is probably involved in the cytokineinduced production of IL-8. Basal production of IL-8 by endothelial cells was inhibited by IFN-γ but not by glucocorticosteroids suggesting that the basal production may be mediated through systems other than NF-κB. In conclusion, this thesis reports that leukocytes can sequester in the pulmonary capillaries due to factors other than adhesion molecules and without affecting the microvasculature. However, when leukocyte sequestration involves IL-8, interfemns and glucocorticostemids might present novel opportunities for therapeutic amelioration of deleterious effects associated with leukocyte sequestration.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2003. , 74 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 770
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-27529Local ID: 12186ISBN: 91-7373-527-2 (print)OAI: oai:DiVA.org:liu-27529DiVA: diva2:248081
Public defence
2003-01-24, Berzeliussalen, Hälsouniversitetet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-24Bibliographically approved
List of papers
1. An isolated blood-perfused guinea-pig lung model for simultaneous registration of haemodynamic, microvascular and respiratory variables
Open this publication in new window or tab >>An isolated blood-perfused guinea-pig lung model for simultaneous registration of haemodynamic, microvascular and respiratory variables
1997 (English)In: Acta Physiologica Scandinavica, ISSN 0001-6772, E-ISSN 1365-201X, Vol. 159, no 4, 293-302 p.Article in journal (Refereed) Published
Abstract [en]

We have developed an optimized isolated lung perfusion system, which possesses several advantages. Firstly, studies of microvascular, respiratory, haematological and biochemical variables are combined in one model. Secondly, blood perfusion resulted in less oedema formation than buffer-perfused lungs, and high Po2 through ventilation with room air. Finally, data for the variables can be displayed, controlled and recorded in real time using a computerized system permitting subsequent processing (e.g. filtering without destroying original data). In this paper we discuss the basic behaviour of the model in terms of vascular resistance, vascular permeability, respiration and neutrophil sequestration. In addition, the effects of oleic acid, histamine and histamine receptor blockers were tested, and two methods of calculating vascular permeability are discussed. The way in which different anaesthetics affect the neutrophil content of lung tissue and blood was also investigated. In the model, oleic acid increased pulmonary vascular resistance and permeability, whereas histamine did not affect either permeability or the pre/postcapillary vascular resistance ratio. However, histamine receptor blockers increased this ratio, indicating that there was endogenous histamine release. The neutrophil content of the isolated lungs was increased, but this did not affect the variables measured. There was also accumulation of neutrophils in the lungs of blood donor animals, due to CO2 sedation. However, CO2 sedation proved to be superior to pentobarbital or ketamine anaesthesia in maintaining the levels of neutrophils circulating in the blood. In conclusion, this model seems to be sensitive and to yield reproducible results regarding the physiology or pathophysiology of the lung.

Keyword
anaesthesia; capillary permeability; histamine; histamine receptor blockers; MPO; oleic acid; pulmonary resistance
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81852 (URN)10.1046/j.1365-201X.1997.00103.x (DOI)
Available from: 2012-09-24 Created: 2012-09-24 Last updated: 2017-12-07Bibliographically approved
2. Leukocyte sequestration in isolated guinea pig lungs during extracorporeal circulation: effects on microvascular function
Open this publication in new window or tab >>Leukocyte sequestration in isolated guinea pig lungs during extracorporeal circulation: effects on microvascular function
2000 (English)In: Blood Purification, ISSN 0253-5068, E-ISSN 1421-9735, Vol. 18, no 2, 121-127 p.Article in journal (Refereed) Published
Abstract [en]

Neutrophils accumulate in patient lungs during clinical hemodialysis and in isolated blood-perfused guinea pig lungs due to the contact between blood and extracorporeal system. However, it is unclear how these sequestered and partly activated neutrophils affect the lung microvasculature. We, therefore, studied pulmonary vascular resistance, vascular permeability, gas exchange, and oxygen free radical production in isolated guinea pig lungs during perfusion with whole blood containing partly ‘activated’ neutrophils in comparison with perfusions using leukopenic blood. We also connected a Cuprophan hemodialysis membrane to the whole-blood perfusion system in order to investigate whether a dialyzer, which may further activate leukocytes, affects lung microvascular permeability, vascular resistances, and reactive oxygen species production. The sequestered neutrophils did not seem to markedly affect the lung microvascular function, since neither the leukocyte-free perfusion nor the hemodialysis membrane altered any of the measured variables as compared with whole-blood perfusion in a system without a dialyzer. We conclude that neutrophils, whether activated by a perfusion system or by a dialysis membrane, can accumulate in isolated lungs without adversely affecting the microvascular function.

Keyword
oxygen free radicals, isolated lung, capillary permeability, hemodialysis
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-49687 (URN)10.1159/000014435 (DOI)10838471 (PubMedID)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2012-09-24Bibliographically approved
3. Corticosteroids and interferons inhibit cytokine-induced production of IL-8 by human endothelial cells
Open this publication in new window or tab >>Corticosteroids and interferons inhibit cytokine-induced production of IL-8 by human endothelial cells
2000 (English)In: Cytokine, ISSN 1043-4666, E-ISSN 1096-0023, Vol. 12, no 4, 355-360 p.Article in journal (Refereed) Published
Abstract [en]

IL-8, secreted by endothelial cells at the site of inflammation, participates in recruitment and transmigration of leukocytes. IL-8 may also have pathophysiological consequences in inflammatory and immunological disorders. We have investigated the effect of interferons (IFNs) and glucocorticosteroids (GCs) on cytokine induced secretion and production of IL-8 by human umbilical endothelial cells (HUVEC). There was a low spontaneous secretion of IL-8 by unstimulated HUVEC which increased after 6 or 24 h of stimulation with the pro-inflammatory cytokines TNF-α or IL-1β. IFN-γ as well as the GCs, Dexamethasone and Budesonide, inhibited TNF-α induced IL-8 secretion in a dose-dependent manner. IFNs may have a general modulating effect, since IFN-α also inhibited the TNF-α-induced IL-8 secretion. There was a slight, but significant, increase in the content of intracellular IL-8 in stimulated HUVEC. However, there was no difference between stimulation with IL-1β or TNF-α alone or in combination with IFNs or GCs, whereas inhibition of IL-8 secretion with monensin increased IL-8 content suggesting that IFNs and GCs inhibit synthesis rather than secretion of IL-8. In conclusion, IFNs or GCs may be useful for inhibiting IL-8 production by endothelial cells and could thus be used for therapeutic modulation of the inflammatory response.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-47672 (URN)10.1006/cyto.1999.0557 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2012-09-24Bibliographically approved
4. Modulation of Cytokine-Induced Production of IL-8 in Vitro by Interferons and Glucocorticosteroids
Open this publication in new window or tab >>Modulation of Cytokine-Induced Production of IL-8 in Vitro by Interferons and Glucocorticosteroids
2004 (English)In: Inflammation, ISSN 0360-3997, E-ISSN 1573-2576, Vol. 28, no 2, 77-88 p.Article in journal (Refereed) Published
Abstract [en]

Interleukin-8 (IL-8) has been implicated in the pathogenesis of inflammation and cancer. Intracellular levels of cytokine-induced IL-8 in human umbilical vein endothelial cells (HUVEC) were modulated using interferons and steroids to further elucidate their mechanism. Basal and cytokine-induced production of IL-8 was studied using a novel ELISA application, flow cytometry, and RT-PCR. The intracellular amount of IL-8 increased after 6-h stimulation with TNF- (30%) or IL-1ß (55%) which was doubled when Golgi transport was disrupted using monensin. IFN-γ decreased the intracellular amount of IL-8 by 60% in both unstimulated and TNF--stimulated cells, but only when secretion was blocked using monensin. Dexamethasone inhibited the TNF--induced production by 33%, but had no effect in unstimulated cells. Our study indicated that both, dexamethasone and IFN inhibit TNF--induced upregulation of IL-8 at the mRNA level. It could be speculated that they inhibit IL-8 production by affecting different gene regulatory mechanisms.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-24058 (URN)10.1023/B:IFLA.0000033023.76110.51 (DOI)3617 (Local ID)3617 (Archive number)3617 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-09-24Bibliographically approved

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