Thèse (Ph. D.)--Université Laval, 2005. / Titre de l'écran-titre (visionné le 28 mars 2007). Bibliogr.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Bibliography: leaves 169-205. This thesis suggests that the testicular macrophage exhibits characteristics similar to that of a suppressor macrophage phenotype. The inhibition of lymphocyte proliferation by the testicular macrophage, its unique cytokine profile, high basal production of GM-CSF and prostaglandins, and the refractoriness to LPS all suggests a role that contributes to the immune privilege that is afforded the testis. However, these aspects of testicular macrophage immuno-biology also support a role in local cell-cell communication and regulation of the normal physiology of the testis, and macrophages may be directly involved in Leydig cell steriogenesis.

<p>The tolerance towards the semi-allogenic foetus is obtained through both systemic and local changes in the maternal immune response. Locally, in the decidua, the cell composition differs from that found in the blood; natural killer (NK) cells and macrophages being the major cell types. Decidual macrophages (dMØ), which are alternatively activated, and trophoblasts, placental cells of foetal origin, are believed to participate in the foetal tolerance at the foetal-maternal interface. To test the recruitment ability of macrophages and trophoblasts, and to test if these cells are responsible for the special cell composition in the decidua, a migration assay was established. In this migration assay peripheral blood mononuclear cells (PBMC) were allowed to migrate through Matrigel-coated transwell inserts into lower wells containing a recruiting stimulus. After testing several conditions, a protocol was established for further use. The results showed that <em>in vitro</em> alternatively activated macrophages, which display many of the surface markers as dMØ, hold a recruiting ability and recruit monocytes. Further there was an indication that trophoblasts also hold a recruiting ability. Neither cell types were shown to recruit NK cells. In conclusion, this study presents a suitable protocol for assessing chemotactic factors and different cell type’s ability to recruit cells from blood. Although the experiments need to be repeated and extended and the recruitment ability of dMØ needs to be evaluated in detail before a final conclusion can be drawn, the preliminary data indicated that macrophages and trophoblasts can recruit monocytes.</p>

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Au cours de ce travail, nous nous sommes attachés à étudier certaines réponses cellulaires secondaires à l’activation des récepteurs purinergiques P2X dans deux modèles différents, les macrophages péritonéaux et les cellules des glandes sous-maxillaires. Ces cellules contribuent à notre immunité innée, soit tournée vers l’intérieur (macrophages), soit vers l’extérieur (glandes sous-maxillaires). Nous avons dans un premier temps confirmé par Western blot et par des dosages de la concentration intracellulaire de calcium ([Ca2+]i) que les deux types de cellules étudiés expriment des récepteurs P2X4 et P2X7 fonctionnels. Nous nous sommes alors concentrés sur deux réponses impliquées dans la protection de l’hôte contre les agressions et l’élimination de pathogènes : la production d’espèces réactives de l’oxygène (ROS) ainsi que la sécrétion de la cytokine pro-inflammatoire interleukine-1beta (IL-1beta). Nos résultats montrent que la production de ROS en réponse à l’ATP extracellulaire est secondaire à l’activation d’une NADPH oxydase dans les deux types de cellules. Cette réponse est médiée par les récepteurs P2X7 ainsi que, dans les macrophages, par d’autres récepteurs purinergiques comme par exemple les récepteurs P2X4 et des récepteurs P2Y. Dans les glandes exocrines, contrairement aux macrophages, la protéine kinase C ainsi que ERK1/2 interviennent dans l’activation de la NADPH oxydase. Par la suite nous avons comparé la régulation de l’expression et de la sécrétion d’IL-1beta par les macrophages et les glandes sous-maxillaires. Nous avons observé que l’IL-1beta est présente dans la salive collectée chez des souris injectées par de la pilocarpine. Des analyses par ELISA, RT-PCR et Western blot montrent que la cytokine est exprimée de manière constitutive par les cellules acineuses et ductales des glandes sous-maxillaires, à un niveau plus élevé que dans les macrophages. Contrairement aux cellules phagocytaires, l’expression de la cytokine dans les cellules des glandes salivaires n’est pas augmentée suite à la stimulation par des lipopolysaccharides. De même, dans ces cellules l’ATP n’a pas provoqué la sécrétion d’IL-1beta malgré l’efflux de K+ secondaire à l’activation des récepteurs P2X7. Dans une dernière série d’expériences nous avons évalué les effets du peptide antimicrobien CRAMP sur les macrophages murins. Le CRAMP a inhibé toutes les réponses secondaires à l’activation des récepteurs P2X7 (ouverture du canal cationique, formation de pore, production de ROS, libération d’IL-1beta, d’acide oléique et de lactate déshydrogénase). L’inhibition par le CRAMP de l’augmentation de la [Ca2+]i en réponse à l’ATP n’était pas médiée par les récepteurs aux peptides formylés car les agonistes de ces récepteurs n’ont pas bloqué cette augmentation. Le CRAMP n’a pas eu d’effet sur l’augmentation de la [Ca2+]i secondaire à l’activation des récepteurs P2X4 par une combinaison d’ATP et d’ivermectine. Nos expériences ont révélé que les récepteurs P2X7 sont couplés à diverses voies de signalisation dans les macrophages et dans les glandes exocrines. Les voies activées diffèrent en fonction du type de cellules. Nous avons également conclu que les peptides antimicrobiens de la famille de cathélicidines ne sont pas des agonistes universels des récepteurs P2X7.

Wear particle-induced inflammation leading to periprosthetic osteolysis remains a major cause of hip implant failure. As polyethylene particles from conventional metal-on-polyethylene implants have been associated with these failures, an interest in lower wear metal-on-metal (MM) bearings has emerged. However, the biological effects of nanometer-size chromium oxide particles, predominant type of wear particles produced by MM implants, remain mostly unknown. Therefore, this study aimed to determine the cytotoxicity of nanometer-size Cr2O3 particles on macrophages in vitro, by analyzing their effects on cell mortality and cytokine release and comparing them with those of similarly-sized alumina (Al2O3) particles (known to be relatively bioinert). Results showed that at high concentrations, nanometer-size Cr2O3 particles can be cytotoxic to macrophages, inducing significant decreases in total cell numbers and increases in necrosis. Results also showed that, at high concentrations, the cytotoxicity of Cr2O3 particles was overall higher than that of Al2O3 particles, even though Cr2O3 and Al2O3 are both stable forms of ceramic materials. However, it appeared to be lower than that of previously reported conventional polyethylene and CoCrMo particles. Therefore, chromium oxide particles may not be the main culprit in initiating the inflammatory reaction in MM periprosthetic tissues.

ABCA1 is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apoA-I. This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e. channel, pump or flippase, remains unknown. In this study we analyzed the extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of relevant non-expressing cells. We found that the extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as WT-ABCA1, is unable to raise extracellular ATP concentration. This suggests a causal relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of elevated extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under the conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e. < μM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane bound ecto-nucleotidase CD39, abolishes cholesterol efflux to apoA-I. Based on these results we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.

The mechanisms of idiosyncratic drug reactions (IDRs) are unknown; however, most appear to be immune-mediated. Their idiosyncratic nature and the paucity of animal models make mechanistic studies very difficult. One of the few animal models is penicillamine-induced autoimmunity in Brown Norway rats. The major focus of this thesis was the use of this model to study the interaction between penicillamine and macrophages, the involvement of Th17 cells, and extension of this model to idiosyncratic drug-induced liver injury. One of the costimulatory signals leading to T cell activation appears to be reversible Schiff-base formation between an amine on T cells and an aldehyde on macrophages. We hypothesized that penicillamine binds to these aldehydes leading to macrophage activation and autoimmunity. By using biotinylated aldehyde-reactive agents such as ARP, we demonstrated the existence of aldehydes on the surface of macrophages. We synthesized biotinylated-penicillamine and it also binds to macrophages. Several proteins to which ARP binds were identified providing clues to the signal transduction pathways leading to macrophage activation. Biological consequences of this binding were investigated with a microarray study. ARP binding was also observed in the macrophage cell line, RAW264.7, and incubation with penicillamine stimulated the production of TNF-α, IL-6, and IL-23. Hydralazine and isoniazid, which are known to cause a lupus-like syndrome in humans and irreversibly bind to aldehyde groups, were also found to activate RAW264.7 cells. Th17 cells are prominent in autoimmune syndromes and Th17-associated cytokines such as IL-17 were elevated in the penicillamine-treated animals that developed autoimmunity. We have hypothesized that some drug-induced liver injury has an autoimmune component. A pilot study quantified serum concentrations of 26 cytokines/chemokines in patients with various forms of acute liver failure (ALF): idiosyncratic drug-induced ALF, acetaminophen-induced ALF, and viral hepatitis. IL-17 was elevated in 60% of patients with idiosyncratic drug-induced ALF, which supports an autoimmune component in these patients; however, it was also elevated in many cases of acetaminophen-induced ALF, presumably released by the innate immune system. These studies provide important insights into the mechanism of penicillamine-, hydralazine-, and isoniazid-induced autoimmunity and also provide clues to other IDRs that may have an autoimmune component.

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