Cellular mechanisms of inflammation— meeting held in Lie ...

Cellular mechanisms of inflammation—meeting held in Liege, Belgium, 12September 1997

Organized by G. CamusCentre Interdisciplinaire de Biochimie de l’Oxygene,Institut de Chimie, B6, Sart Tilman, 4000 Liege,BelgiumTel: (+32) 4 36 63 360Fax: (+32) 4 36 62 866

Ramipril prevents endothelial dysfunctioninduced by oxidized LDL

G. Berkenboom, I. Langer, A. El Ajani,Y. Carpentier and J. FontaineCardiology Department, Erasme Hospital,Route de Lennik, 808, 1070 Brussels, Belgium

Oxidized (ox) LDL plays a key role in hyper-cholesterolaemia-induced impairment of endothe-lium-derived (ED) NO-mediated responses. To assesswhether long-term ACE inhibition may prevent this Edysfunction, four groups of rats were treated inparallel; Group 1 (Ram 10) received ramipril 10 mg/kg/day p.o. for 6 weeks; Group 2 (Ram + HOE)received ramipril 10 mg/kg/day p.o. for 4 weeks andthe last 2 weeks a cotreatment with ramipril plusHOE140 (a bradykinin B2 antagonist 500 m g/kg/day,subcutaneous injections); Group 3 (Ram 1) receivedramipril 1 mg/kg/day p.o. for 6 weeks; Group 4 servedas control. Rings of thoracic aorta from these groupswere contracted by phenylephrine (Phe) and relaxa-tions to acetylcholine (Ach, 10 nM to 0.1 mM) weredetermined in the absence (oxLDL–) and in thepresence (oxLDL+) of human Cu2+ -oxidized LDL(300 m g/ml, 30 min preincubation).

Groups Maximalresponse (%)

oxLDL– oxLDL+

Area under curve

oxLDL– oxLDL+

Ram 10 (n = 9) 97 ± 2* 93 ± 5 358 ± 19* 369 ± 22Ram + HOE (n = 9) 82 ± 3 48 ± 3§ 284 ± 10 165 ± 9§Ram 1 (n = 12) 91 ± 4* 76 ± 7§ 289 ± 20 250 ± 32Control (n = 12) 82 ± 4 53 ± 5§ 269 ± 15 202 ± 17§

(mean ± SEM); % of Phe-induced plateau; *P <0.05 vs. control; §P<0.05 vs. oxLDL–.

The toxic effect of oxLDL is directly related to the NOsynthase pathway as L-arginine (0.1 mM) coincubatedwith oxLDL completely prevented the E dysfunction incontrol group. Aortic cyclicGMP content, reflectingNO availability, was significantly increased in Ram 10(by 76% vs control) but not in the other groups. Thus,long-term ACE inhibition dose-dependently protectsagainst the acute toxicity of oxLDL; the mechanism isbradykinin-dependent and seems to involve an upregu-lation of EDNO synthase pathway via B2 kininreceptor.

Endotoxaemia and production of TNF-ainduced by strenuous physical exercise inmanG. Camus, M. Nys, J. Poortmans, I. Venneman,T. Monfils, G. Deby-Dupont, A. Juchmes-Ferir,C. Deby, M. Lamy and J. DuchateauFNRS, Center for the Biochemistry of Oxygen,Institute of Chemistry, Sart Tilman, Universityof Liege, Belgium

To examine whether endotoxaemia accompanyingstrenuous physical work is involved in exercise-induced increase in plasma tumour necrosis factoralpha (TNF-a ), 14 male recreational athletes [meanage: 28 ± 1 (SEM) years] were studied. Exerciseconsisted of a 1.5 km river swim, a 40 km bicycle race,and a 10 km road race. Mean time to complete the racewas 149.8 ± 4.8 (± SEM) min (range: 128–195 min).The plasma concentrations of TNF-a were significantlyhigher than baseline values immediately after exerciseand 1 h later (P < 0.001). This variable returned to pre-race levels the day after exercise. Marked, transientdecreases in the plasma levels of anti-lipopolysacchar-ide (LPS) IgG and IgM antibodies primarily directedagainst a panel of Gram-negative LPSs were observedafter the race. An inverse, highly significant relation-

0962-9351/98/010053-05 © $9.00 1998 Carfax Publishing Ltd 53

Conference abstracts

Mediators of Inflammation, 7, 53–57 (1998)

ship between the increase in plasma TNF-a concentra-tions and the changes in circulating anti-LPS IgMantibodies was found (r = – 0.7; P < 0.01). This findingstrongly argues for the involvement of endotoxaemiain the exercise-induced production of TNF-a observedin our subjects.

Interleukin-10 treatment oflipopolysaccharide-activated macrophagesimproves control of Trypanosoma cruziinfection by enhancing nitric oxide releaseF. Jacobs,1 D. Chaussabel,1 C. Truyens,2V. Leclerq,1 Y. Carlier,2 M. Goldman1

and B. Vray1

1Laboratoire d’Immunologie Experimentale,Faculte de Medecine, Universite Libre deBruxelles, route de Lennik 808, 1070 Brussels;2Laboratoire de Parasitologie, Faculte deMedecine, Universite Libre de Bruxelles,Belgium

Trypano soma cruzi, a protozoan parasite, is theaetiological agent of Chagas’ disease. Murine macro-phages infected by T. cruzi trypomastigotes, releasenitric oxide (NO) when suitably activated. Severalstudies have indicated the involvement of NO in thecontrol of T. cruzi infection. Interleukin 10 (IL-10) isa polyfunctional cytokine with various properties andproduced by several cell types including macro-phages. To date, relationships between IL-10, macro-phage activation by lipopolysaccharide (LPS) andT.cruzi infection are still unclear. In this study, wedemonstrated that, in LPS-activated and T. cruzi-infected macrophages, the addition of murine recom-binant IL-10 increased the NO production and resul-ted in a better control of T. cruzi infection. Inaddition, endogenous IL-10 produced by T. cruzi-infected mouse peritoneal macrophages participate inthe NO production since neutralization of endoge-nous IL-10 by specific monoclonal antibodies inhibitsNO release. Taken together, these data highlight theactivating role of IL-10 on LPS-activated macrophagesthrough a NO-mediated control of T. cruzi infection.

Critical role of reactive oxygenintermediates (ROI) in TNF-a and IL-10production after liver cold ischaemia andreperfusion in miceO. Le Moine, H. Louis, P. Stordeur, J. M. Collet,M. Goldman and J. DeviereDepartments of Gastroenterology andImmunology, Hospital Erasme. ULB, Brusselsand Laboratory of Organic Chemistry, Facultede Medecine, Universite de Mons-Hainaut,Belgium

ROI, cytokines and adhesion molecules are releasedand expressed by the liver during experimental and

human liver transplantation. They play a role in livercell damage and in the recruitment of inflammatoryand immunocompetent cells that might eventuallylead to graft dysfunction and rejection. To date, thestimuli leading to cytokine release are unknown. Theaim of the present study was to assess the role of ROI incytokine release by the ischaemic liver after reperfu-sion and its modulation by exogenous administrationof IL-10. In that setting we developed an endotoxin-free isolated perfused mouse liver system. Liver injurywas assessed by the release of ALT in the liver effluent.Cytokines were measured by commercially availablekits and mRNA by RT-PCR. After increasing coldischaemic times, both TNF, IL-10 and IFN-g are releasedafter reperfusion of the organ (peak levels after75 min). At the gene level, mRNA expression for thesecytokines was increased only after reperfusion. Twoantioxidants, N-acetylcysteine (NAC) and allopurinolsignificantly decreased TNF and IL-10 release (– 64%, –62% and –49%, –57%, respectively), as did an inhibitorof the transcription factor NF-kB mobilization (– 73%and – 76% for TNF and IL-10, respectively). Liver injurywas also significantly decreased by these compounds.In addition, non-ischaemic livers submitted to anoxidant stress by hydrogen peroxide released bothTNF and IL-10, an effect abolished by concomitantinfusion of NAC. Finally, recombinant IL-10 (rIL-10)added during reperfusion of ischaemic livers was ableto abolish TNF and IFN-g release and decrease liverinjury. In conclusion, ROI are involved in cytokinerelease after reperfusion of ischaemic livers. Bothantioxidants, inhibitors of NF-kB mobilization and rIL-10 are able to dampen cytokine production and liverinjury. This may represent a new therapeutic modalityin transplantation medicine.

Protection of alveolar type II cells byperflubron against mediators ofinflammation

M. Mathy-Hartert,1 G. Deby-Dupont,1,2

C. Deby1 and M. Lamy1,2

1Centre for the Biochemistry of Oxygen, Institutde Chimie, B6a, Sart Tilman, 4000, Liege;2University Hospital Centre, University ofLiege, Sart Tilman, 4000, Liege, Belgium

Perfluorocarbons (PFC) are liquids with high O2

dissolving capacity and low surface tension. Theseproperties led to the utilization of PFC for liquidventilation in lung pathologies such as acute respira-tory distress syndrome (ARDS) where PFC supportedgas exchange and reduced surface tension at thealveolar air liquid interface. ARDS is accompanied byacute inflammatory process, where excessive poly-morphonuclear (PMN) activation is responsible forlung injury. We investigated the biocompatibility of

Abstracts

54 Mediators of Inflammation · Vol 7 · 1998

perfluorooctylbromide (PFOB) with cultured humanepithelial alveolar cells (A549 line). Confluent cellswere overlayed with PFOB, covered with culturedmedium and incubated at 37°C for time periodsranging from 1 h to 17 h. Incorporation of PFOB intothe cells was investigated by gradient density cen-trifugation and a cytotoxicity index (CI) was esti-mated from the release of pre-incorporated 51Cr. Noincorporation of PFOB was obtained (cell density wasnot increased) and no cytotox icity of PFOB wasobserved. To test the cytoprotective effects of PFOBagainst mediators of inflammation, PMN were isolatedfrom whole blood and activated by phorbol myristateacetate (PMA) (10–6 M). PFOB-overlayed cells werecovered with HBSS + albumine 0.1 % containingsupernatant of activated PMN or activated PMN andwere incubated for 3 h at 37°C. A cell ratio (PMN/A549) of 10 was used. Control incubations wereperformed with alveolar cells w ithout PFOB. Super-natant of activated PMN induced a weak cytotoxicity(CI = 24.4 ± 5.4%) which was largely reduced in thepresence of PFOB (CI = 8.6 ± 3.2%). Activated PMNinduced a higher cytotoxicity (CI = 78.0 ± 8.3%)which was not significantly reduced by PFOB (CI =72.4 ± 7.2%). In conclusion, PFOB was innocuous forcells and did not enter the cells. PFOB protected thealveolar cells against oxidative stress induced by thesupernatant of activated PMN, but could not effica-ciously protect against a large number of stimulatedPMN.

Antioxidant activity of propofol:chemiluminescence study andcytoprotection

M. Mathy-Hartert,1 G. Deby-Dupont,1 P. Hans2

and M. Lamy2

1Centre de Biochimie de l’Oxygene, Universitede Liege B6a Sart Tilman 4000 Liege; 2Serviced’Anesthesie-Reanimation, CHU, B35 SartTilman 4000 Liege, Belgium

The anaesthetic agent propofol (PPF) has beenreported to possess an in vitro antioxidant activity asefficient as that of a -tocopherol (vitamin E). Westudied the effects of PPF on in vitro lipoperoxidationinduced by three free radical systems (hydroxyl, ferryland oxo-ferryl radicals). Lipoperoxidation was quanti-fied by gas chromatography measurement of pentanereleased from linoleic acid decomposition. We alsoseparately examined the effects of PPF, Diprivan®(the commercial form of the drug) and intralipid (IL)(the vehicle of PPF in Diprivan®) on the luminolenhanced chemiluminescence (CL) of stimulatedhuman neutrophils or endothelial cells. Finally, wetried the effects of Diprivan® in the protection ofendothelial cells against oxidant stress induced by the

myeloperoxidase/H2O2/Cl– system. PPF inhibitedlipoperoxidation in a dose-dependent manner, and itsantioxidant activity was similar to that of vitamin E.Both compounds were however more active againsthydroxyl radical induced lipoperoxidation. The CL of5 3 105 polymorphonuclear neutrophils (PMN)stimulated by 10–6 M phorbol myristate acetate (PMA)was inhibited similarly by Diprivan® and IL, but notin a dose-dependent manner. On the contrary, PPFinhibited CL in a dose dependent manner: 37.3 ± 6.3% inhibition was obtained with 5 3 10–5 M PPF, a dosecorresponding to a clinically relevant concentration.The CL of 5 3 105 endothelial cells was also inhibitedby PPF, Diprivan®, and to a lesser extent by IL. Theseantioxidant properties of PPF were correlated withprotection of endothelial cells submitted to an oxi-dant stress. PPF thus exerts a high antioxidantcapacity, that cannot impair the intracellular phago-cytic cell functions of PMN because PPF did not enterinto the cells (HPLC measurement after extraction).

Ceftazidime reduces free radicalsformation by alveolar cells submitted toanoxia and reoxygenation

A. Mouithys-Mickalad,1 M. Mathy-Hartert,1G. Deby-Dupont,1,2 C. Deby1 and M. Lamy1,2

1Centre for the Biochemistry of Oxygen,Institute of Chemistry, B6a, DomaineUniversitaire du Sart Tilman, 4000 Liege;2Department of Anaesthesiology and IntensiveCare, Centre Hospitalier Universitaire, B35,Domaine Universitaire du Sart Tilman, 4000,Liege, Belgium

Cultured cells have been shown to generate activeoxygen species (AOS) during anoxia-reoxygenationsequence. Electron spin resonance (ESR) spectro-scopy with alpha-(4-pyridyl-l-oxide)-N-tert-butylni-trone (4-POBN) spin trapping, in the presence ofethanol, was used to monitor over time the AOS,especially lipid-derived free radicals, formed by cul-tured human alveolar type II cells (A549) exposed (at37°C) to anoxia (N2 , l h) and reoxygenenation (O2 , orunder ambient air in HBSS). We investigated theeffects of ceftazidime (CAZ), a third generationcephalosporin antibiotic which was demonstrated toprotect endothelial cells submitted to NaOCl-oxidantstress,1 to inhibit lipid peroxidation2 and desactivatesinglet oxygen.3 We also compared the effects of CAZwith those of superoxide dismutase (SOD), TIRON(4,5-dihydroxy-1, 3-benzene-disulfonic acid) a specificreagent for O2

–o scavenging, and L-NMMA (an NOsynthase inhibitor). The reaction between AOS or lipidradicals and ethanol leads to ethoxy free radicals,which then react w ith 4-POBN to form specific spinadducts (POBN-EtOH). Anoxied alveolar cells pro-

Cellula r Mechanisms o f inflammation

Mediators of Inflammation · Vol 7 · 1998 55

duced AOS. This production increases with thereoxygenation time and the ESR spectra show that thefollowing hyperfin splittings obtained from experi-mental ESR spectra (aN = 15.7 G and aH = 2.7 G) arein agreement with the data of literature.4 When SOD(200 U/ml) was added before anoxia-reoxygenation,the ESR signal was decreased (37% of inhibition)versus control (alveolar cells alone). Likewise, the ESRsignal intensity was reduced when cells were incu-bated in the presence of either CAZ (2 mM; 37% ofinhibition) or TIRON (0.1 mM; 67% of inhibition) aswell as L-NMMA (1 mM; 43% of inhibition). TIRONcould be a better inhibitor because it enters inside thecells. CAZ and L-NMMA do not enter the cells andreact with free radicals outside. Our ESR resultsdemonstrate that CAZ thus reduces the free radicalformation, confirming the quenching effect on AOSwe recently reported.3,4

References1. Mathy-Hartert M, e t a l. Mediato rs Inflamm 1995; 4 : 437–443.2. Mathy-Hartert M, e t a l. Inten sive Care Med 1994; 5 (2suppl): 74.3. Deby-Dupont e t al. Bio chem Biophy s Acta (1997).4. Connor HD, et a l. Transplantation 1992; 54: 199–204.

Technical development towards themicrodialysis monitoring of cytokines inthe brain extracellular space duringischaemic insult

V. Olislagers,1 S. Goldman,2 M. A. Laute,1S. Blecic,1 M. Goldman3 and S. Dethy1

1Laboratoire de Neurochime Clinique (Servicede Neurologie); 2Unite TEP-CyclotronBiomedical; 3Service d’Immunologie,Universite Libre de Bruxelles-Hopital Erasme,808 route de Lennik, 1070 Bruxelles, Belgium

TNF-a has unpredictable neurodestructive or neu-roprotective effects on the ischaemic neuron. Tobetter define the influence of cytokines during brainischaemia, we should access the kinetics of theirconcentration in the brain extracellular space (EC)where they interact with neuronal and glial receptors.The microdialysis (MD) technique allows such an ECmonitoring of bioactive molecules. To test the MDtechnique for the detection of TNF-a EC changes inresponse to brain insult, we have developed a modelof LPS injection in the rat striatum. Two MD probes(CMA 12, 100 kDa) were inserted in the right (LPS)and left (PBS) striatum. Previous experiments haveshown that TNF-a EC concentration determined bythis technique in intact rat striatum (no injection) was100 pg/ml. Samples were collected every 60 min for7 h and TNF-a levels were determined by ELISA. MDdemonstrated progressive increase of TNF-a EC con-

centration in the right striatum from 5 to 7 h after LPSinjection (n = 10, ANOVA P < 0.0001) reaching amaximum value of 666 ± 239 pg/ml at 7 h post-injection. This value was 120% higher than in thecontrol PBS-injected striatum. The technique will nowbe applied to the monitoring of TNF-a EC concentra-tion in the rat brain after ischaemic insult producedby middle cerebral artery occlusion.

Plasmatic myeloperoxydase (MPO) level inhorses with strangulated intestinalobstruction

D. Serteyn,1 S. Grulke,1 H. Benbarek,1M. Mathy-Hartert,2 G. Deby-Dupont,2 C. Deby2

and M. Lamy2

1Anesthesiologie Generale, Clinique etPathologie Chirurgicale des Grands Animaux,Universite de Liege, B41 4000 Sart Tilman;2Centre de Biochimie de l’Oxygene, Universitede Liege, B6a 4000 Sart Tilman, Belgium

In equine pathology, mortality associated with intesti-nal strangulation in horses usually ranges between50% and 80%. The intestinal injury that developsduring strangulation may progress after detorsion dueto irreversible cellular alterations that occurred dur-ing the ischaemic period, or due to exacerbation ofcellular injury as a result of the cascade of eventsinitiated on reperfusion. Myeloperoxidase is a specificazurophilic granule enzyme from neutrophils. Itproduces oxidant species that can induce highcellular toxicity. It is released in plasma whenneutrophils degranulate and is thus a w itness ofneutrophil activation. We used an original specifictechnique (radioimmunoassay) for measurement ofthe levels of myeloperoxidase in horses operated forintestinal obstruction; 15 horses were operated for alarge intestine obstruction: nine horses with non-strangulated obstruction (control group) and six withstrangulated obstruction leading to severe complica-tion and death (strangulated group). MPO was meas-ured at reception (P1), at time of surgery andintensive care (P2), at the phase of recuperation orcomplication (P3). Mean MPO values were higher instrangulated group for the three phases: 243 ng/ml atP1, 426 ng/ml at P2 and 379 ng/ml at P3 compared to122, 169 and 107 ng/ml for P1, P2 and P3 respectivelyin control group (P < 0.01 for P2 and P3 between thetwo groups).

The significant difference of plasmatic MPO valuesdemonstrated a neutrophil activation that could beexplained by an endotoxin release by the ischaemicbowel. This high leukocyte activation could thuscontribute to the development of an ox idative stressduring equine acute abdomen.

Abstracts

56 Mediators of Inflammation · Vol 7 · 1998

Reactive oxygen intermediates increasethe production of IL-8 by human dendriticcells

V. Verhasselt, C. Buelens, F. Willemsand M. GoldmanLaboratory of Experimental Immunology,U.L.B., Campus Erasme, 808, route de Lennik,1070 Brussels, Belgium

Reactive oxygen intermediates (ROI) are commonlyproduced during inflammatory processes as well asduring postischaemic reperfusion. Besides their cyto-toxic effects, ROI also affect cell functions bymodifying their redox potential and/or by activatingtranscription factors. We studied the effects of H202,a major source of ROI, on human dendritic cells

(DC). Dose–response experiments showed that theoptimum concentration of H202 was 100 m M. At thisconcentration, H202 stimulated the production ofhigh concentrations of IL-8 (6 ng/ml) and increasedbut to a lesser extent the production of TNF-a(0.4 ng/ml) by DC. H202 did not affect their produc-tion of IL-6, IL-12 and IL-10. When we looked at theeffects of H202 on the expression of surface mole-cule of DC, we showed that H202 moderatelyincreased their expression of HLA-DR and decreasedtheir expression of B7–1 and CD40. H202 did notmodify significantly neither B7–2 nor CD54 expres-sion. From these data, we might extrapolate that DCwhich play critical role in the induction of specificimmune responses are also involved in inflammationby amplifying the recruitment of neutrophilsthrough IL-8 synthesis.

Cellula r Mechanisms o f inflammation

Mediators of Inflammation · Vol 7 · 1998 57

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