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L'efferocytose est un phénomène physiologique par lequel des millions de cellules apoptotiques sont efficacement éliminées par phagocytose sans provoquer une réaction inflammatoire. L'efficacité de ce processus nécessite une interaction rapide entre la cellule apoptotique et son phagocyte afin d'éviter l'entrée de la première dans une phase de nécrose secondaire. Cette interaction implique des motifs de la surface des 2 cellules qui peuvent interagir directement ou aussi indirectement via des molécules de pontage. Ce dernier rôle est associé à C1q, premier composant du système du complément. En effet, C1q favorise l'élimination des cellules apoptotiques et aussi la réponse tolérogène en interagissant avec des ligands présents de part et d'autre de la synapse efferocytaire. La calréticuline (CRT) de surface fait partie de ces ligands. Initialement connue comme co-récepteur, avec CD91, de la queue collagène de C1q à la surface du phagocyte, la CRT est aujourd'hui décrite comme un signal « eat-me » de la surface des cellules apoptotiques où elle peut aussi interagir avec les têtes globulaires de C1q. Des études récentes ont soulevé le potentiel immunogénique de la CRT, notamment au cours de la mort des cellules cancéreuses. Ainsi, la CRT de surface joue un rôle important dans l'efferocytose même si l'on ne sait pas exactement comment cette protéine chaperonne résidente du réticulum endoplasmique est exposée à la membrane plasmique. Dans un premier temps, j'ai démontré que la CRT augmente à la surface des cellules Jurkat rapidement après l'induction de l'apoptose, à un stade où la phosphatidylsérine, marqueur emblématique de l'apoptose, n'est pas encore détectée. D'une manière intéressante, C1q est capable d'interagir directement avec la CRT exposée à la surface des cellules à ce stade « pré-apoptotique », et de favoriser significativement leur phagocytose par les macrophages THP1. Dans un deuxième temps, j'ai mis en évidence la présence de la CRT dans le milieu extracellulaire et montré qu'elle varie avec l'évolution de l'apoptose. De plus, la CRT soluble est capable d'induire la migration des macrophages THP1, d'augmenter l'expression à leur surface de CD14, récepteur impliqué dans l'efferocytose, et de stimuler la macropinocytose, un processus utilisé par les phagocytes lors de la phagocytose des cellules apoptotiques. Cela suggère que la CRT extracellulaire peut moduler la biologie du phagocyte. Enfin, la CRT exogène peut se lier à la surface des macrophages et peut être ainsi une source externe de la CRT retrouvée à la surface des phagocytes. / Efferocytosis is a physiological phenomenon whereby millions of apoptotic cells are efficiently removed by phagocytosis without inducing an inflammatory response. The efficiency of this process requires rapid interaction between the apoptotic cell and the phagocyte in order to prevent the entry of the dying cell in a secondary necrosis phase. This interaction involves patterns of the surface of the 2 cells that can interact directly or indirectly via bridging molecules. The latter role is associated to C1q, the first component of the complement system. Indeed, C1q promotes the removal of apoptotic cells and the tolerogenic response by interacting with ligands present on either side of the efferocytic synapse. Surface exposed calreticulin (CRT) is one of these ligands. Initially known as the co-receptor, with CD91, to the collagenous tail of C1q on the phagocyte surface, CRT is now described as an “eat-me" signal of the apoptotic cell surface where it can also interact with the globular heads of C1q. Recent data have revealed the immunogenic potential of CRT, especially in the case of cancer cell death. Thus, surface exposed CRT plays an important role in efferocytosis even if it is not fully understood how this reticulum endoplasmic resident protein gets to the plasma membrane. I firstly demonstrated that CRT increases rapidly on the surface of Jurkat cells after the induction of apoptosis, at a stage where phosphatidylserine, emblematic marker of apoptosis, is not yet detected. Interestingly, C1q is capable of interacting directly with this “pre-apoptotic” cell surface exposed CRT, and promotes significantly the uptake of Jurkat cells by THP1 macrophages at this stage. Secondly, I demonstrated the presence of CRT in the extracellular medium whose content depends on the evolution of apoptosis. Furthermore, soluble CRT induces the migration of THP1 macrophages, increases their surface expression of CD14, a receptor involved in efferocytosis, and stimulates macropinocytosis, a process used by phagocytes during phagocytosis of apoptotic cells. These results suggest that the extracellular CRT can modulate the biology of the phagocyte. Finally, exogenous CRT binds to the surface of macrophages and can therefore be an external source of the CRT found on the phagocyte surface.

Thesis (doctoral)--Utrecht, 1979.

Orientador: Flávio Henrique Caetano / Banca: Ana Maria Costa Leonardo / Banca: Maeli Dal Pai Silva / Banca: Maria Tercilia Vilela de Azeredo Oliveira / Banca: Carmem Silvia Fontanetti Christofoletti / Resumo: O presente estudo teve como objetivo analisar os leucócitos circulantes em microscopia de luz e eletrônica e sua distribuição sazonal, além de procurar estabelecer o período de renovação celular desses leucócitos, e principalmente de caracterizar os fagócitos mononucleares do sangue de tartaruga, e sua capacidade de fagocitose frente a material inerte. Neste trabalho utilizou-se seis tartarugas Phrynops hilarii, originárias de ilhas do estuário do rio Guaíba Porto Alegre (RS), que estavam ambientadas em nosso biotério. A coleta de sangue foi realizada em todos os períodos sazonais, por punção de vasos laterais do pescoço e coletados em tubos de ensaio heparinizados. Foram realizados esfregaços sanguíneos, corados com Leishmann e Giemsa, contando-se quinhentas células de cada animal e após a obtenção dos dados, foi aplicado o teste estatístico de Bonferroni. Para a análise autorradiográfica foi injetado 1000μCi / kg de thymidine-H A. Para microscopia eletrônica processamos a nata leucocitária obtida por meio de centrifugação do sangue, para a analise citoquímica incubamos com citidina-5'-monofosfato, betaglicerofosfato de sódio, Trimetafosfatase, para averiguar a resposta fagocitária utilizamos 0,01% de carvão coloidal.Os resultados mostram que os leucócitos de Phrynops hilarii tem descrição de leucócitos semelhantes às outras espécies, somente os basófilos e linfócitos não sofreram alterações em sua distribuição sazonal. Todos os leucócitos com exceção dos basófilos apresentaram renovação celular após sete dias. Caracterizamos monoblasto, promonócito, monócitos e macrófago no sangue circulante bem como a capacidade dos fagócitos mononucleares de fagocitar células mortas e materiais inerte. / Abstract: The aim of this study was to analyze the leukocytes in the blood using electronic and light microscopy and their seasonal distribution, also to characterize the leukocyte cells replacement and mainly to characterize the mononuclear phagocytes in the blood and their phagocytic capacity. In this study, it was used six turtles (Phrynops hilarii), caught at the Guaíba river estuary, Porto Alegre, Rio Grande do Sul, Brazil and lodged for 1 week at the Central Animal House, University of São Paulo, Ribeirão Preto, São Paulo, Brazil. Blood was obtained during the seasonal periods by puncturing the lateral vessels of the neck. The blood samples were stained by Leishmann and Giemsa, counting five hundred cells in each animal. After the obtained data, it was applied the Bonferroni test as statistical method. For the autoradiographic analysis, it was injected in the circulating blood 1000 μCi/kg of 3Hthymidine. For electronic microscopy, it was processed the leukocyte substrate by circulating blood centrifugation. For cytochemical analyses, blood smears were air dried, post-fixed in 4% formalin and submitted to the determination of the following enzyme activities: acid phosphatases (β-g1ycerophosphatase and citidine-5′-sodium monophosphatase), and trimetaphosphatase. The results showed that the leukocytes of Phrynops hilarii have the leukocytes description similar to the other species, only the basophiles and lymphocytes did not suffer alterations in their seasonal distribution. All the leukocytes, in exception of the basophiles showed cells replacement after seven days. It was characterized the monocytes and macrophages in the circulating blood as well as the phagocytes capacity. / Doutor

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-11Bitstream added on 2014-06-13T20:21:25Z : No. of bitstreams: 1 pitol_dl_dr_rcla.pdf: 2375764 bytes, checksum: 1ce362e81dd4a2ee6f04869e84243e98 (MD5) / O presente estudo teve como objetivo analisar os leucócitos circulantes em microscopia de luz e eletrônica e sua distribuição sazonal, além de procurar estabelecer o período de renovação celular desses leucócitos, e principalmente de caracterizar os fagócitos mononucleares do sangue de tartaruga, e sua capacidade de fagocitose frente a material inerte. Neste trabalho utilizou-se seis tartarugas Phrynops hilarii, originárias de ilhas do estuário do rio Guaíba Porto Alegre (RS), que estavam ambientadas em nosso biotério. A coleta de sangue foi realizada em todos os períodos sazonais, por punção de vasos laterais do pescoço e coletados em tubos de ensaio heparinizados. Foram realizados esfregaços sanguíneos, corados com Leishmann e Giemsa, contando-se quinhentas células de cada animal e após a obtenção dos dados, foi aplicado o teste estatístico de Bonferroni. Para a análise autorradiográfica foi injetado 1000μCi / kg de thymidine-H A. Para microscopia eletrônica processamos a nata leucocitária obtida por meio de centrifugação do sangue, para a analise citoquímica incubamos com citidina-5'-monofosfato, betaglicerofosfato de sódio, Trimetafosfatase, para averiguar a resposta fagocitária utilizamos 0,01% de carvão coloidal.Os resultados mostram que os leucócitos de Phrynops hilarii tem descrição de leucócitos semelhantes às outras espécies, somente os basófilos e linfócitos não sofreram alterações em sua distribuição sazonal. Todos os leucócitos com exceção dos basófilos apresentaram renovação celular após sete dias. Caracterizamos monoblasto, promonócito, monócitos e macrófago no sangue circulante bem como a capacidade dos fagócitos mononucleares de fagocitar células mortas e materiais inerte. / The aim of this study was to analyze the leukocytes in the blood using electronic and light microscopy and their seasonal distribution, also to characterize the leukocyte cells replacement and mainly to characterize the mononuclear phagocytes in the blood and their phagocytic capacity. In this study, it was used six turtles (Phrynops hilarii), caught at the Guaíba river estuary, Porto Alegre, Rio Grande do Sul, Brazil and lodged for 1 week at the Central Animal House, University of São Paulo, Ribeirão Preto, São Paulo, Brazil. Blood was obtained during the seasonal periods by puncturing the lateral vessels of the neck. The blood samples were stained by Leishmann and Giemsa, counting five hundred cells in each animal. After the obtained data, it was applied the Bonferroni test as statistical method. For the autoradiographic analysis, it was injected in the circulating blood 1000 μCi/kg of 3Hthymidine. For electronic microscopy, it was processed the leukocyte substrate by circulating blood centrifugation. For cytochemical analyses, blood smears were air dried, post-fixed in 4% formalin and submitted to the determination of the following enzyme activities: acid phosphatases (β-g1ycerophosphatase and citidine-5′-sodium monophosphatase), and trimetaphosphatase. The results showed that the leukocytes of Phrynops hilarii have the leukocytes description similar to the other species, only the basophiles and lymphocytes did not suffer alterations in their seasonal distribution. All the leukocytes, in exception of the basophiles showed cells replacement after seven days. It was characterized the monocytes and macrophages in the circulating blood as well as the phagocytes capacity.

<p>Apoptosis, or programmed cell death, is a controlled process by which aged or damages cells are eliminated in multicellular organisms. Neutrophils, short-lived phagocytes of the innate immune system, are highly equipped effectors that can sense, locate, ingest and kill bacterial pathogens. Inflammatory mediators and the presence of bacterial products at the foci of infection regulate the function and life span of these cells. Modulation of neutrophil apoptosis and the subsequent clearance by scavenger cells, such as macrophages, is part of a balanced inflammatory process leading to resolution of inflammation. Many pathogens are capable of modulating host cell apoptosis, and thereby influence the progression of disease. Hence, this thesis was aiming at elucidating mechanisms involved in pathogen- and host-modulated apoptosis and its contribution to the inflammatory process.</p><p>We found that different routes of bacterial entry, i.e. through invasion or by receptor-mediated phagocytosis, triggered different signaling pathways within phagocytes. Invasion of virulent Salmonella caused apoptosis, a process requiring activation of the Rho GTPases Rac1 and Cdc42. On the other hand, phagocytosis of the non-invasive Salmonella inhibited apoptosis despite similar intracellular survival as the invasive bacteria. Protection against phagocytosis-induced apoptosis was regulated by tyrosine- and PI3-kinase-dependent activation of AKT (also called PKB for protein kinase B). Furthermore, inhibiting the intraphagosomal production of reactive oxygen species (ROS) in neutrophils during phagocytosis of <em>E. coli</em> decreased apoptosis below spontaneous apoptosis, further indicating that both pro- and anti-apoptotic pathways are triggered by receptor-mediated phagocytosis.</p><p>Type 1 fimbria-expressing <em>E. coli </em>adhering to neutrophils resisted ingestion, and induced a ROS-dependent apoptosis by a cooperative effect of the FimH adhesin and LPS. To explore how compartmentalization of ROS during neutrophil activation was involved in modulating apoptosis, we evaluated the stability of lysosomes. In contrast to phagocytosis of <em>E. coli</em>, the adhesive strain induced intracellular non-phagosomal ROS production which triggered early permeabilization and release of lysosomal enzymes to the cytosol. Cathepsin B and/or L were responsible for targeting of the pro-apoptotic Bcl-2 protein Bid, thereby inducing mitochondrial damage, and apoptosis. These data propose a novel pathway for ROS-induced apoptosis in human neutrophils, where the location of the ROS rather than production <em>per se</em> is important.</p><p>Moreover, we found that pathogen-induced apoptotic neutrophils, in contrast to uninfected apoptotic neutrophils, activated blood-monocyte derived macrophages to increase their FcγRI surface expression and to produce large quantities of the pro-inflammatory cytokine TNF-α. This demonstrates that during the early phase of infection, pathogen-induced neutrophil apoptosis will help local macrophages to gain control over the microbes. Furthermore, we suggest that heat shock protein 60 and 70 represent a stress signal that enables macrophages to distinguish between, and react differently to, uninfected and inflammatory apoptotic neutrophils.</p>

Apoptosis, or programmed cell death, is a controlled process by which aged or damages cells are eliminated in multicellular organisms. Neutrophils, short-lived phagocytes of the innate immune system, are highly equipped effectors that can sense, locate, ingest and kill bacterial pathogens. Inflammatory mediators and the presence of bacterial products at the foci of infection regulate the function and life span of these cells. Modulation of neutrophil apoptosis and the subsequent clearance by scavenger cells, such as macrophages, is part of a balanced inflammatory process leading to resolution of inflammation. Many pathogens are capable of modulating host cell apoptosis, and thereby influence the progression of disease. Hence, this thesis was aiming at elucidating mechanisms involved in pathogen- and host-modulated apoptosis and its contribution to the inflammatory process. We found that different routes of bacterial entry, i.e. through invasion or by receptor-mediated phagocytosis, triggered different signaling pathways within phagocytes. Invasion of virulent Salmonella caused apoptosis, a process requiring activation of the Rho GTPases Rac1 and Cdc42. On the other hand, phagocytosis of the non-invasive Salmonella inhibited apoptosis despite similar intracellular survival as the invasive bacteria. Protection against phagocytosis-induced apoptosis was regulated by tyrosine- and PI3-kinase-dependent activation of AKT (also called PKB for protein kinase B). Furthermore, inhibiting the intraphagosomal production of reactive oxygen species (ROS) in neutrophils during phagocytosis of E. coli decreased apoptosis below spontaneous apoptosis, further indicating that both pro- and anti-apoptotic pathways are triggered by receptor-mediated phagocytosis. Type 1 fimbria-expressing E. coli adhering to neutrophils resisted ingestion, and induced a ROS-dependent apoptosis by a cooperative effect of the FimH adhesin and LPS. To explore how compartmentalization of ROS during neutrophil activation was involved in modulating apoptosis, we evaluated the stability of lysosomes. In contrast to phagocytosis of E. coli, the adhesive strain induced intracellular non-phagosomal ROS production which triggered early permeabilization and release of lysosomal enzymes to the cytosol. Cathepsin B and/or L were responsible for targeting of the pro-apoptotic Bcl-2 protein Bid, thereby inducing mitochondrial damage, and apoptosis. These data propose a novel pathway for ROS-induced apoptosis in human neutrophils, where the location of the ROS rather than production per se is important. Moreover, we found that pathogen-induced apoptotic neutrophils, in contrast to uninfected apoptotic neutrophils, activated blood-monocyte derived macrophages to increase their FcγRI surface expression and to produce large quantities of the pro-inflammatory cytokine TNF-α. This demonstrates that during the early phase of infection, pathogen-induced neutrophil apoptosis will help local macrophages to gain control over the microbes. Furthermore, we suggest that heat shock protein 60 and 70 represent a stress signal that enables macrophages to distinguish between, and react differently to, uninfected and inflammatory apoptotic neutrophils.

Made available in DSpace on 2018-06-27T18:56:28Z (GMT). No. of bitstreams: 3 Valnir de Paula.pdf: 2329033 bytes, checksum: 989bd5d1109793d5b0ba4e36a71ad934 (MD5) Valnir de Paula.pdf.txt: 111221 bytes, checksum: e193938f84364f16942c08f8c27a80b8 (MD5) Valnir de Paula.pdf.jpg: 3313 bytes, checksum: b9abaf2a3f9be69ba98dca42617b7fef (MD5) Previous issue date: 2011-12-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The use of nanoscale products in several areas, including medicine, is a growing reality. The magnetic nanoparticles (MNPs) for being able to present a significant effect of magnetization when exposed to an external magnetic field, have been the focus of several studies, and among their applications is their use as contrast agent in magnetic resonance images. This technique provides images based on the nuclear behavior of the atoms of anatomical structures, which can be best highlighted by the use of contrast agents, usually paramagnetic. The MNPs represent an alternative to the current class of paramagnetic contrast agents for nuclear magnetic resonance, used for a long time, with advantages from a physical point of view, because they highlight even more the magnetic behavior of protons in different tissues, especially liver, spleen and lymphatic system, whose defense cells endocyte these MNPs, making healthy parenchyma dark (hyposignal), so that any injuries stand out in the images, facilitating their identification. This study has aimed to assess the contrast degree of the organs of the phagocyte system, injecting NPMs doses ranging from 0.46 to 7.2 mg/kg in rats, by caudal intravenous flow and subjecting them to nuclear magnetic resonance imaging. The MNPs was divided into two groups, both with a core of magnetite, varying the coating, which has been dextran in group 1, and oleic acid in group 2. The expected effect was that the organs of the phagocyte system would have some degree of signal loss in the images, indicating that the NPMs were internalized by the cells of these organs. With the usual contrast agent, paramagnetic, which does not enter cells, the effect is the hypersignal in the vascular system and in hypervascularized organs. We have compared the images obtained from T1 TSE and T2 TSE sequences with the control obtained before injection. The results have shown that both dextran coated MNPs and the ones coated with oleic acid have caused the hyposignal effect in the images, ranging from weak to strong, depending on the administered dose, especially in T2 TSE sequences. The dextran coated MNPs have shown higher efficiency, considering that the hyposignal effects have occurred with lowers doses, compared to the effects caused by NPMs coated with oleic acid. It can be concluded, given the evident hyposignal effect presented by the organs of the phagocyte system, the potential application of MNPs as a contrast agent in magnetic resonance studies. / A utilização de produtos de escala nanométrica nas mais diversas áreas, inclusive na medicina, é uma realidade crescente. As nanopartículas magnéticas (NPMs), por serem capazes de apresentar efeito expressivo de magnetização quando expostas a um campo magnético externo, têm sido foco de vários estudos e, entre as suas aplicações está o uso como agente de contraste em imagens de ressonância magnética nuclear. Esta técnica fornece imagens baseadas no comportamento nuclear dos átomos das estruturas anatômicas, as quais podem ser melhor destacadas pelo uso de agentes de contraste. As NPMs representam uma classe alternativa aos atuais agentes de contraste, paramagnéticos, com vantagens do ponto de vista físico, pois destacam ainda mais o comportamento magnético dos prótons de diferentes tecidos. Isto é mais evidente no fígado, baço e sistema linfático, cujas células de defesa endocitam estas NPMs, tornando o parênquima sadio escuro (hipossinal), de forma que eventuais lesões se sobressaeam nas imagens, facilitando a sua identificação. O objetivo deste trabalho foi avaliar o grau de contrastação dos órgãos do sistema fagocitário, injetando-se doses de NPMs que variaram de 0,46 a 7,2 mg/Kg em ratos, por via endovenosa caudal e submetendo-os a imageamento por ressonância magnética nuclear. As NPMs foram divididas em 2 grupos, ambos com núcleo de magnetita, variando-se o revestimento, que no grupo 1 foi de dextrana e o do grupo 2, de ácido oléico. O efeito desejado foi que os órgãos do sistema fagocitário apresentassem algum grau de perda de sinal nas imagens, indicando que as NPMs foram internalizadas pelas células desses órgãos. Com o agente de contraste usual, paramagnético, que não entra nas células, o efeito é de hiperssinal no sistema vascular e em órgãos hipervascularizados. Comparou-se as imagens obtidas de sequências T1 TSE e T2 TSE com as de controle, obtidas antes da injeção. Os resultados obtidos evidenciaram que, tanto as NPMs revestidas com dextrana, quanto às revestidas com ácido oléico causaram efeito de hipossinal nas imagens, que variaram de fraco a acentuado, dependendo da dose administrada, principalmente em sequências T2 TSE. As NPMs revestidas com dextrana apresentaram maior eficiência, considerando que os efeitos de hipossinal ocorreram com doses menores do que as revestidas com ácido oléico. Pode-se concluir, considerando o evidente efeito de hipossinal apresentado pelos órgãos do sistema fagocitário, que há potencial de aplicação destas NPMs como agente de contraste em estudos de ressonância magnética.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24515号 / 医博第4957号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 森信 暁雄, 教授 竹内 理, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM