Les médiateurs lipidiques de l’inflammation dont le leucotriène B4 (LTB4) et le facteur d’activation plaquettaire (PAF) permettent la régulation de la migration des neutrophiles polymorphonucléaires (PMNs) et l’extravasation plasmatique au site inflammatoire. Afin de déterminer leurs rôles dans la régulation de la migration des PMNs au site inflammatoire, nous avons étudié leur effet potentiellement coopératif en utilisant une approche pharmacologique à l’aide d’antagonistes sélectifs des récepteurs du LTB4 et du PAF dans un modèle d’inflammation dermique chez le lapin. Les résultats montrent un effet inhibiteur additif des antagonistes des deux médiateurs lipidiques, lorsque utilisés de façon concomitante, sur la migration des neutrophiles induite par le LTB4, le PAF et aussi sur des médiateurs non-chimiquement apparentés comme le facteur nécrosant des tumeurs (TNFα), ainsi que sur l'inhibition de l’extravasation plasmatique induite par le leucotriène D4, suggérant un rôle régulateur des récepteurs du LTB4 et du PAF dans la migration des PMNs au site inflammatoire. Nous avons déterminé le rôle de ces médiateurs dans la régulation de la migration des PMNs en réponse à une ischémie-reperfusion des membres inferieurs chez le lapin. Les résultats appuient l’hypothèse selon laquelle le LTB4 et le PAF exercent un rôle important dans l’accumulation des PMNs au site inflammatoire. En effet l’administration concomitante des antagonistes des récepteurs de ces deux médiateurs lipidiques a réduit de façon significative la migration des PMNs aux poumons, intestins et foie. Nos résultats contribuent à élucider le rôle du LTB4 et du PAF dans la régulation de l’extravasation des PMNs et du plasma au site inflammatoire. / Inflammatory lipid mediators including leucotriene B4 (LTB4) and platelet activating factor (PAF) regulate the trafficking of polymorphonuclear neutrophils (PMNs) and plasma extravasation at inflammatory sites. To delineate their role in regulating PMNs extravasation, we studied the effect of PAF and/or LTB4 selective receptor antagonists in dermal inflammation induced by a variety of agonists in a rabbit bioassay model. The results show that there is an additive inhibitory effect when the two antagonists are used concomitantly on PMNs dermal accumulation induced by LTB4 and PAF, as well by chemically unrelated agonists including TNFα, in addition to inhibiting plasma extravasation induced by LTD4. These results support a regulatory role of LTB4 and PAF in regulating PMNs trafficking and plasma extravasation at inflammatory sites. Next, we studied the regulatory role of lipid mediators in regulating PMNs trafficking in response to hind limb ischemia-reperfusion. The results show that the administration of both PAF and LTB4 receptor antagonists reduced significantly PMNs migration to the lung, the liver and the intestine. Our results contributed to elucidate the role of LTB4 and PAF in the regulation of PMNs migration and oedema formation at inflammatory sites.

Made available in DSpace on 2016-06-02T20:18:15Z (GMT). No. of bitstreams: 1 3931.pdf: 5591864 bytes, checksum: b4873ba462675328d3fc2005b8032afb (MD5) Previous issue date: 2011-02-28 / Osteoarthritis (OA), a degenerative joint disease, is one of the most frequent causes of pain in the musculoskeletal system and of the inability to work in Brazil and the world. It is a multifactor, chronic disease, leading to progressive functional inability. It can arise as a result of injuries to structures such as the anterior crossed ligament and/or meniscus (post-traumatic OA), which, in this case, can affect individuals in any age range. The development of osteoarthritis includes multiple changes in the extracellular cartilage matrix, altering the normal morphological configuration of the joint involved, leading to a lack of equilibrium between the synthesis and degradation of products in this matrix. Although OA is not considered primordially as an inflammatory disease, inflammation of the joint has been shown to be a potential amplifier of the degenerative process. Thus the objective of the present study was to analyze potential biological markers in the serum and synovial fluid, and then correlate them with one another and with the morphological, inflammatory and functional alterations found in individuals with chronic injury of the anterior crossed ligament (ACL). The following techniques were used in the study: zymography, to determine the activity of the metallopeptidases 2 and 9 (MMP-2 and MMP-9); an immune-enzymatic assay (ELISA) to determine the presence of systemic and local cytokines; and a manual count of inflammatory cells (mononuclear and polymorphonuclear) by optical microscopy and spectrophotometry, in order to analyze for sulfated glycosaminoglycans (GAGs). The results indicated joint and systemic inflammation in chronic injury of the ACL by the detection of systemic and local cytokines, by the activity of MMP-9 and by the inflow of neutrophils. There were interactions between systemic and local cytokines, in which a cytokine did not always exert the same function in the serum as in the synovial fluid. The interleucines (IL) connected to degradation of the cartilage in chronic injury of the ACL were IL-12, IL-6 and IL-8, and those connected to pain and loss of function were IL-6 and IL-9. In counterpart, MMP-2 showed a negative correlation with the damage to the cartilage. It was concluded that the molecules studied had potential as biomarkers, since alterations were suggestive of injury and degradation of the cartilage. In addition, after the traumatic event resulting in rupture of the ACL, the ambient remained chronically inflamed and this inflammation was crucial for the high index of posttraumatic OA. / A osteoartrite (OA), doença articular degenerativa, é uma das causas mais freqüentes de dor do sistema músculo-esquelético e de incapacidade para o trabalho no Brasil e no mundo. É uma doença crônica, multifatorial, que leva a uma incapacidade funcional progressiva. Pode surgir em decorrência de lesões em estruturas como ligamento cruzado anterior e/ou meniscos (OA pós-traumática), e neste caso, pode afetar indivíduos em qualquer faixa etária. O desenvolvimento da osteoartrite inclui múltiplas mudanças na matriz extracelular da cartilagem, o que altera a configuração morfológica normal da articulação envolvida, levando a um desequilíbrio entre a síntese e degradação dos produtos desta matriz. Apesar da OA não ser considerada primordialmente como uma doença inflamatória, a inflamação articular tem demonstrado um potencial amplificador do processo degenerativo. Sendo assim, o objetivo deste trabalho foi analisar potenciais marcadores biológicos no soro e no líquido sinovial, e em seguida correlacioná-los uns com os outros e com as alterações morfológicas, inflamatórias e funcionais encontradas em sujeitos com lesão crônica do ligamento cruzado anterior (LCA). Para este estudo foram utilizadas técnicas de: zimografia, para verificar a atividade das metalopeptidases 2 e 9 (MMP-2 e MMP-9); Ensaio imunoenzimático (ELISA), para constatar a presença das citocinas sistêmicas e locais; contagem manual de células inflamatórias (mononucleares e polimorfonucleares) por microscopia óptica e espectrofotometria para a análise dos glicosaminoglicanos sulfatados (GAGs). Os resultados apontaram para uma inflamação articular e sistêmica na lesão crônica do LCA, pela detecção de citocinas sistêmicas e locais, pela atividade das MMP-9 e pelo influxo de neutrófilos. Houve interações entre citocinas sistêmicas e locais, nas quais nem sempre uma citocina exerce a mesma função no soro e no líquido sinovial. As interleucinas (IL) ligadas à degradação da cartilagem na lesão crônica do LCA foram IL-12, IL-6 e IL-8 e as ligadas à dor e a perda de função foram IL-6 e IL-8. Em contrapartida, a MMP-2 apresentou correlação negativa com os danos na cartilagem. Conclui-se que, as moléculas estudadas apresentam potencial como biomarcadores, sendo suas alterações sugestivas de lesão e degradação da cartilagem. E ainda, que após o evento traumático responsável pelo rompimento do LCA, o ambiente permanece inflamado cronicamente e que esta inflamação é crucial para o alto índice de OA pós-traumática.

La leptospirose est une zoonose causée par les bactéries du genre Leptospira. Elle touche près de 1 million d'individus par an dans le monde entier et sévit à l'état endémique dans les pays au climat tropical tel que La Réunion. Les manifestations habituelles sont variables d'un individu à l'autre et englobent une simple fièvre jusqu'aux défaillances poly-viscérales avec mortalité dans 5 à 10% des cas. Sa physiopathologie est encore mal comprise, en particulier la part que joue une réponse immunitaire inappropriée dans la genèse des manifestations graves qui surviennent en quelques heures, et avant la mise en place d'une réponse immunitaire adaptative propre à éliminer le microorganisme. Si l'échappement de la bactérie au système du complément est bien documenté, le rôle des acteurs cellulaires du système immunitaire inné reste à étayer. Notre étude avait donc pour objectif d'explorer l'immunopathologie de la leptospirose humaine dans la phase initiale de l’infection. Notre méthodologie s'est appuyée principalement sur des analyses quantitatives et qualitatives des acteurs cellulaires du système immunitaire inné à partir de prélèvements sanguins en phase précoce de la maladie, et comparaison avec la phase de convalescence et des sujets contrôles. Dans un premier temps nous avons montré qu'une population particulière de lymphocytes T impliquée dans la réponse immune innée, les lymphocytes Tγδ, avaient un taux abaissé et que cette baisse était corrélée à la charge bactérienne ainsi qu'à l’intensité de l'atteinte hépatique classiquement retrouvée lors de la leptospirose. Dans un deuxième temps, nous avons analysé les polynucléaires neutrophiles circulants dont le taux augmente d’autant plus que la maladie est sévère, mais sans pour autant présenter de modification de certains marqueurs d’activation ou de recrutement tissulaire (CD15, CD11b, CD182). Une des principales chimiokines des neutrophiles, l'interleukine 8, était à taux peu élevés. Les derniers travaux concernent les principales formes solubles issues des molécules membranaires impliquées dans le processus de recrutement/diapédèse leucocytaire. Nous retrouvons de manière isolée une forte élévation des formes solubles d'E-sélectine et ICAM-1 qui sont notamment exprimées par les cellules endothéliales. Ces augmentations n'étaient pas corrélées aux marqueurs de gravité de la maladie. La signification biologique de cette élévation n’est pas encore connue lors de la leptospirose. L'ensemble de nos données permet d’apporter des informations nouvelles sur des acteurs du système immunitaire inné présents dans le compartiment vasculaire lors de la leptospirose humaine. Cette réponse immunitaire semble inadaptée pour permettre une clairance du pathogène au stade de dissémination hématogène. / Leptospirosis is a bacterial zoonosis caused by Leptospira and affecting 1 million people each year worldwide and mainly in tropical areas such as Reunion Island. Usual presentations encompass flu-like syndrome to multiorgan failure with mortality rate between 5 to 10%. To date, pathophysiology in humans is poorly understood, notably the capacity of innateimmunity to mount a robust response to clear pathogen or to induce tissue damages and contributing to disease severity. Our study aimed at assessing the role of innate immune cells and molecules within the first days of leptospiral infection.Using blood samples, we performed quantitative and qualitative assessment of circulating innate immune cells from leptospirosis cases and healthy controls. The first study explored the levels of gamma-delta T-cells (γδT-cells), a subset of unconventional T cells with innate immune functions. Gamma-delta T cells were found deeply decreased and levels wereinversely correlated to bacterial burden and liver damage. The second study focused on membrane bound receptors indicative of activation and tissue migration ability of neutrophil polymorphonuclear cells: CD15, CD11b, and CD182. Although neutrophil rates were high in leptospirosis cases, the levels of studied receptors were either lower (CD15) or identical to healthy controls (CD11b, CD182). In addition, only low levels of interleukin-8, a key chemokine for neutrophils, was detected in patients. Lastly, we ascertained the plasmatic levels of several shed cell adhesion molecules notably expressed by endothelial cells. The levels of soluble E-selectin and ICAM-1 were significantly increased compared to controls, while P-selectin level was lower. We did not find any correlation with disease severity or organ failure. This finding indicates that endothelial cell may be activated but further experiments are warranted to explain the functional impact of our findings. Altogether, our results add to the field of knowledge of leptospirosis pathophysiology, and in particular the implication of key innate immune cells at the stage of plasmatic bacterial dissemination. Our findings will support the view that there is an inappropriate immune response to Leptospira.

β2-glicoproteína I (β2GPI) é encontrada (200µg/mL) no plasma, 60% livre e 40% em lipoproteínas. Esta proteína de fase aguda, com afinidade por superfícies negativas pode ser clivada pela plasmina. Fragmentos são purificados como dímeros ou multímeros de β2GPI. Formas monomérica e dimérica de β2GPI foram purificadas de soro humano e identificadas por SDS-PAGE, imunoblot e ELISA Somente a forma monomérica foi detectada no teste ELISA Os efeitos de ambas as formas sobre o burst respiratório de polimorfonucleares humanos (PMN), estimulados in vitro com zymosan opsonizado, foram estudados por quimioluminescência amplificada por luminol ou lucigenina e citometria de fluxo, pela oxidação do OCFH. A forma monomérica inibiu a quimioluminescência amplificada por luminol (-43.6 x -7.33 AEU/s), mas não por lucigenina, e aumentou a oxidação de DCFH e a produção de Óxido Nítrico (•NO). É provável que o •No, via peroxinitrito, medeie os efeitos de β2GPI sobre o burst respiratório de PMN. / Circulating blood contains approximately 200µg/mL of β 2-glycoprotein I (β2GPI), either free (60%) or lipoprotein bound (40%). This acute phase protein, with affinity for negative surfaces, can be cleaved by plasmin. Fragments purify as dimeric or multimeric (β2GPI. Both (β2GPI forms were purified from human sera and identified by SDS-PAGE, immunoblotting and ELISA. ELISA reactivity was dependent on the monomeric status of (β2GPI. The effects of dimeric and monomeric (β2GPI upon respiratory burst of human polimorphonuclear neutrophils (PMN) stimulated in vitro with opsonized zymosan were studied. Respiratory burst was evaluated by luminol- or lucigenin-amplified chemiluminescence, or by DCFH oxidation (flow-cytometry assay). The monomeric, but not the dimeric form, inhibited the luminol chemiluminescence of zymosan-stimulated PMNs (-43.6 x -7.33 AEU/s). Lucigenin chemiluminescence was insensitive to (β2-GPI. Monomeric (β2GPI increases both DCFH oxidation and nitric oxide production. Nitric oxide, probably through peroxynitrite reactions, mediates (β2GPI effects upon PMNs respiratory burst.

β2-glicoproteína I (β2GPI) é encontrada (200µg/mL) no plasma, 60% livre e 40% em lipoproteínas. Esta proteína de fase aguda, com afinidade por superfícies negativas pode ser clivada pela plasmina. Fragmentos são purificados como dímeros ou multímeros de β2GPI. Formas monomérica e dimérica de β2GPI foram purificadas de soro humano e identificadas por SDS-PAGE, imunoblot e ELISA Somente a forma monomérica foi detectada no teste ELISA Os efeitos de ambas as formas sobre o burst respiratório de polimorfonucleares humanos (PMN), estimulados in vitro com zymosan opsonizado, foram estudados por quimioluminescência amplificada por luminol ou lucigenina e citometria de fluxo, pela oxidação do OCFH. A forma monomérica inibiu a quimioluminescência amplificada por luminol (-43.6 x -7.33 AEU/s), mas não por lucigenina, e aumentou a oxidação de DCFH e a produção de Óxido Nítrico (•NO). É provável que o •No, via peroxinitrito, medeie os efeitos de β2GPI sobre o burst respiratório de PMN. / Circulating blood contains approximately 200µg/mL of β 2-glycoprotein I (β2GPI), either free (60%) or lipoprotein bound (40%). This acute phase protein, with affinity for negative surfaces, can be cleaved by plasmin. Fragments purify as dimeric or multimeric (β2GPI. Both (β2GPI forms were purified from human sera and identified by SDS-PAGE, immunoblotting and ELISA. ELISA reactivity was dependent on the monomeric status of (β2GPI. The effects of dimeric and monomeric (β2GPI upon respiratory burst of human polimorphonuclear neutrophils (PMN) stimulated in vitro with opsonized zymosan were studied. Respiratory burst was evaluated by luminol- or lucigenin-amplified chemiluminescence, or by DCFH oxidation (flow-cytometry assay). The monomeric, but not the dimeric form, inhibited the luminol chemiluminescence of zymosan-stimulated PMNs (-43.6 x -7.33 AEU/s). Lucigenin chemiluminescence was insensitive to (β2-GPI. Monomeric (β2GPI increases both DCFH oxidation and nitric oxide production. Nitric oxide, probably through peroxynitrite reactions, mediates (β2GPI effects upon PMNs respiratory burst.

Les polynucléaires neutrophiles représentent 50-70% des leucocytes sanguins et possèdent un rôle majeur dans la défense de l’organisme contre les pathogènes. Le Ca2+ est un second messager qui joue un rôle primordial dans le chimiotactisme, la phagocytose, la dégranulation et la production de formes réactives de l’oxygène (FRO) afin de neutraliser l’agent pathogène. Dans ces cellules, l’influx calcique de type SOCE est essentiel pour l'homéostasie calcique. Il est peu étudié en raison du manque d’outils pharmacologiques spécifiques d’où l’importance dans un premier temps de chercher de nouvelles molécules. Les cellules T Jurkat dont le SOCE est largement caractérisé servent de modèle pour la caractérisation initiale de ces molécules. Le 2-APB est parmi les molécules les plus largement utilisées dans la caractérisation du SOCE en raison de sa double activité sur le SOCE avec une potentialisation à [1-10 μM] et une inhibition à [> 20 μM]. En revanche, ce produit manque de spécificité et agit sur d’autres cibles cellulaires comme les récepteurs à l’inositol (1,4,5)-trisphosphate (InsP3Rs). La 1ère étape est de sélectionner à partir d’analogues commerciaux du 2-APB (Methoxy-APB, Dimethoxy-APB, Cyclic-APB, Benzothienyl-APB, Thienyl-APB et MDEB), des composés plus spécifiques et également plus efficaces que la molécule mère. Deux molécules se sont distinguées : le MDEB comme uniquement potentialisant du SOCE et le Benzothienyl-APB comme un puissant inhibiteur. En revanche, tous les analogues du 2-APB inhibent les InsP3Rs à l’exception du MDEB qui semble plus spécifique du SOCE. L’effet du MDEB sur le courant calcique, ICRAC, a été étudié grâce à la technique du patch-clamp. Il augmente d’environ 4 fois l’amplitude de ICRAC par rapport à celle enregistrée dans les cellules contrôle. Par ailleurs, le MDEB ralentie l’inactivation rapide de ICRAC due au Ca2+. Sur le plan physiologique, le MDEB à des concentrations croissantes inhibe la synthèse de l’IL-2 dans les cellules Jurkat stimulées et ceci malgré son effet potentialisant du SOCE. Cette activité est liée à son effet pro-apoptotique dans les cellules Jurkat stimulées. Le MDEB et le Benzothienyl-APB caractérisés dans la 1ère partie nous ont servi d’outils potentiels afin d’étudier le SOCE des cellules PLB-985 différenciées en cellules proches de neutrophiles. Le SOCE a été induit soit par un traitement des cellules avec la thapsigargine (Tg) soit de manière physiologique avec les peptides fMLF et le WKYMVm deux chimioattractants, ligands des récepteurs aux peptides formylés FPR et FPRL1 respectivement. En plus, le SOCE induit par la Tg est modulable par le 2-APB, potentialisé par le MDEB et inhibé par le Benzothienyl-APB. La phagocytose des levures par les cellules PLB-985 différenciées ainsi que la production de FRO intraphagosomales ont été inhibées par le MDEB et le Benzothienyl-APB. Les FRO extracellulaires ont été également inhibées par Benzothienyl-APB en revanche à cause de la forte interférence du MDEB avec la technique de mesure nous n’avons pas pu étudier ses activités. En conclusion, le MDEB et le Benzothienyl-APB sont de nouveaux outils pharmacologiques potentialisant ou inhibant le SOCE des leucocytes, qui nous permettront dans l’avenir une meilleure compréhension de l'entrée calcique et ses rôles dans ces cellules. / Neutrophils represent 50-70% of human blood leukocytes; their role is to protect the body against pathogens. Calcium is a second messenger which plays an important role in chemotaxis, phagocytosis, degranulation and the production of reactive oxygen species (ROS) in order to eliminate microbes. In neutrophils, the mechanism of store-operated calcium entry (SOCE) is essential for calcium homeostasis. However, neutrophil SOCE is not well understood because of the lack of specific pharmacological tools. It is necessary to first identify and characterize new molecules using a model of Jurkat T cells in which SOCE was the best characterized. 2-APB is the most widely used molecule in SOCE characterization due to its dual activity with a potentiation at lower concentrations [1-10 μM] and an inhibition at higher concentrations [> 20 μM]. However, this molecule lacks specificity because it acts on other cellular targets such as inositol (1,4,5)-trisphosphate receptors (InsP3Rs). The first step is to select from a library of 8 commercial 2-APB analogues (Methoxy-APB, Dimethoxy-APB, Cyclic-APB, Benzothienyl-APB, Thienyl-APB and MDEB) those that are more specific and also more efficient molecules than 2-APB. Two interesting molecules were identified, MDEB as the only SOCE potentiating product currently known and the Benzothienyl-APB, which is a strong inhibitor. Like 2-APB, all these analogues inhibit InsP3Rs except MDEB, which seems to be more specific. The effect of MDEB on the calcium current, ICRAC, was also studied using the patch-clamp technique. MDEB increases ~4 times the ICRAC amplitude in comparison with control. Otherwise, MDEB slows down the fast Ca2 +-dependent inactivation of ICRAC. Functionally, MDEB at increasing concentrations inhibits IL-2 synthesis in stimulated Jurkat T cells despite its potentiating activity on SOCE. The inhibition is due to MDEB induced apoptosis in stimulated Jurkat T cells. MDEB and Benzothienyl-APB were then used as tools to study SOCE in a neutrophil-like cell model, the differentiated PLB-985 cells. SOCE was induced either by treatment of cells with thapsigargin (Tg) or physiologically with the chemotactic peptides fMLF and WKYMVm, ligands of formyl peptide receptors FPR and FPRL1 respectively. In addition, Tg-induced SOCE was modulated by 2-APB, potentiated by MDEB and inhibited by Benzothienyl-APB. The consequences of these analogues on neutrophil functions were also studied. Phagocytosis of yeasts by PLB-985 cells and intraphagosomal ROS production were inhibited by MDEB and Benzothienyl-APB. Furthermore, extracellular ROS were also inhibited by Benzothienyl-APB. However, because of the high interference of MDEB with our techniques, its activities could not be studied. In conclusion, MDEB and Benzothienyl-APB are new analogues of 2-APB potentiating and inhibiting SOCE, which allow us in the future a better understanding of leukocyte SOCE and its cellular roles.