RESUMO
PURPOSE: The hypothesis that locally-released iloprost, a synthetic prostacyclin analog, affects macrophage phenotype at a microdialysis implant in the subcutaneous space of rats was tested. Macrophage activation towards alternatively-activated phenotypes using pharmaceutical release is of interest to improve integration of implants and direct the foreign body reaction toward a successful outcome. METHODS: Macrophage cell culture was used to test iloprost macrophage activation in vitro. Microdialysis sampling probes were implanted into the subcutaneous space of Sprague-Dawley rats to locally deliver iloprost in awake- and freely-moving rats. Monocyte chemoattractant protein -1 (CCL2) was quantified from collected dialysates using ELISA. Immunohistochemical staining was used to determine the presence of CD163+ macrophages in explanted tissues. RESULTS: Iloprost reduced CCL2 concentrations in NR8383 macrophages stimulated with lipopolysaccharide. CCL2 concentrations in collected dialysates were similarly reduced in the presence of iloprost. Iloprost caused an increase in CD163+ cells in explanted tissue surrounding implanted microdialysis probes at two days post probe implantation. CONCLUSIONS: Localized delivery of iloprost caused macrophage activation at the tissue interface of a microdialysis subcutaneous implant in rat. This model system may be useful for testing other potential macrophage modulators in vivo.
Assuntos
Quimiocina CCL2/metabolismo , Iloprosta/química , Iloprosta/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Liberação Controlada de Fármacos , Humanos , Iloprosta/administração & dosagem , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Microdiálise , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/metabolismo , Distribuição TecidualRESUMO
Macrophage activation is of interest in the biomaterials field since macrophages with an M(Dex) characteristic phenotype, i.e., CD68(+)CD163(+), are believed to result in improved integration of the biomaterial as well as improved tissue remodeling and increased biomaterial longevity. To facilitate delivery of a macrophage modulator, dexamethasone-21-phosphate (Dex), microdialysis probes were subcutaneously implanted in male Sprague-Dawley rats. Dex localized delivery was delayed to the third day post implantation as a means to alter macrophage activation state at an implant site. To better elucidate the molecular mechanisms associated with M(Dex) macrophage activation, CCL2 was quantified in dialysates, gene expression ratios were determined from excised tissue surrounding the implant, histological analyses, and immunohistochemical analyses (CD68, CD163) were performed. Delayed Dex infusion resulted in the up-regulation of IL-6 at the transcript level in the tissue in contact with the microdialysis probe and decreased CCL2 concentrations collected in dialysates. Histological analyses showed increased cellular density as compared to controls in response to delayed Dex infusion. Dex delayed infusion resulted in an increased percentage of CD68(+)CD163(+), M(Dex), macrophages in the tissue surrounding the microdialysis probe as compared to probes that served as controls.
Assuntos
Quimiocina CCL2/imunologia , Citocinas/imunologia , Dexametasona/análogos & derivados , Implantes de Medicamento/administração & dosagem , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Dexametasona/administração & dosagem , Relação Dose-Resposta a Droga , Glucocorticoides/administração & dosagem , Fatores Imunológicos/administração & dosagem , Masculino , Microdiálise/instrumentação , Microdiálise/métodos , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/imunologiaRESUMO
Microdialysis sampling probes were implanted into the subcutaneous space on the dorsal side of male Sprague Dawley rats to locally deliver dexamethasone-21-phosphate (Dex) with the aim of altering in vivo macrophage polarization. Macrophage polarization is of significant interest in the field of biomaterials since wound-healing macrophages are a possible means to extend implant life as well as improve tissue remodeling to an implant. Quantitative analysis of CCL2 in collected dialysates, gene expression and immunohistochemistry performed on the tissue surrounding the microdialysis implant were used to evaluate if Dex polarized macrophages. Dex infusion down-regulated IL-6 and CCL2 gene expression and decreased CCL2 concentrations in dialysates collected at the implant site. Dex appeared to have no significant effect on the gene regulation of CD163, a commonly used M2c macrophage surface marker; Arg2; and iNOS2. However, Dex infusion was effective at increasing the number of CD163(+) cells surrounding the implanted microdialysis probe. This work demonstrates the use of microdialysis sampling to deliver agents such as Dex to alter macrophage polarization in vivo while allowing the ability to collect cytokines in the surrounding microenvironment.
Assuntos
Quimiocina CCL2/metabolismo , Dexametasona/análogos & derivados , Macrófagos/efeitos dos fármacos , Microdiálise , Animais , Dexametasona/administração & dosagem , Macrófagos/citologia , Macrófagos/metabolismo , Reação em Cadeia da Polimerase , RatosRESUMO
Microdialysis sampling is a commonly used technique for collecting solutes from the extracellular space of tissues in laboratory animals and humans. Large molecular weight solutes can be collected using high molecular weight cutoff (MWCO) membranes (100kDa or greater). High MWCO membranes require addition of high molecular weight dextrans or albumin to the perfusion fluid to prevent fluid loss via ultrafiltration. While these perfusion fluid additives are commonly used during microdialysis sampling, the tissue response to the loss of these compounds across the membrane is poorly understood. Tissue reactions to implanted microdialysis sampling probes containing different microdialysis perfusion fluids were compared over a 7-day time period in rats. The base perfusion fluid was Ringer's solution supplemented with either bovine serum albumin (BSA), rat serum albumin (RSA), Dextran-70, or Dextran-500. A significant inflammatory response to Dextran-70 was observed. No differences in the tissue response between BSA and RSA were observed. Among these agents, the BSA, RSA, and Dextran-500 produced a significantly reduced inflammatory response compared to the Dextran-70. This work demonstrates that use of Dextran-70 in microdialysis sampling perfusion fluids should be eliminated and replaced with Dextran-500 or other alternatives.
Assuntos
Soluções para Diálise/toxicidade , Reação a Corpo Estranho/etiologia , Microdiálise/efeitos adversos , Tela Subcutânea/efeitos dos fármacos , Animais , Dextranos/metabolismo , Dextranos/toxicidade , Soluções para Diálise/metabolismo , Desenho de Equipamento , Reação a Corpo Estranho/metabolismo , Soluções Isotônicas/metabolismo , Soluções Isotônicas/toxicidade , Masculino , Microdiálise/instrumentação , Microdiálise/métodos , Peso Molecular , Ratos Sprague-Dawley , Solução de Ringer , Fatores de Risco , Soroalbumina Bovina/metabolismo , Soroalbumina Bovina/toxicidade , Tela Subcutânea/metabolismo , Fatores de TempoRESUMO
Multiple checkpoints regulating finely balanced death-versus-survival decisions characterize both thymic development and peripheral homeostasis of T lymphocytes. While exploring the mechanisms of T cell death involved at various stages during the life of a T cell, we have observed and reported a variety of non-redundant roles for apoptosis inducing factor (Aif), a mitochondrial flavoprotein. Aif is ubiquitously expressed in all cell lineages and functions as an NADH oxidase in its mitochondrial location. It is released following the mitochondrial death signals, whereupon it translocates to the nucleus, binds to DNA and causes large-scale DNA fragmentation. During T cell development, Aif is important for developing thymocytes to navigate the double negative (DN)3 to DN4 transition (beta-selection), via its oxidoreductase property which protects the rapidly proliferating cells from death due to reactive oxygen species (ROS). In peripheral mature T cells, Aif deficiency leads to an increased susceptibility of T cell blasts to activation induced cell death (AICD), possibly mediated by its antioxidant function, and decreased sensitivity to neglect-induced death (NID). Thus, Aif seems to have pro-apoptotic and anti-apoptotic roles in the same lineage in different contexts and at different stages. Surprisingly, in the closely related B lymphocyte lineage, Aif deficiency does not result in any abnormality. These findings generate the possibility of specific T cell dysfunction in human disease caused by Aif deficiency, as well as in mitochondriopathies due to other causes. Also, these data raise questions regarding the basis of lineage-specific consequences of the dysfunction/deficiency of apparently ubiquitous molecules.
Assuntos
Fator de Indução de Apoptose/metabolismo , Diferenciação Celular/genética , Linfócitos T/metabolismo , Timo/crescimento & desenvolvimento , Antioxidantes/metabolismo , Apoptose/genética , Fator de Indução de Apoptose/genética , Linhagem da Célula/imunologia , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Linfócitos T/imunologia , Timo/metabolismoRESUMO
Apoptosis-inducing factor (Aif) is a mitochondrial flavoprotein that regulates cell metabolism and survival in many tissues. We report that aif-hypomorphic harlequin (Hq) mice show thymic hypocellularity and a cell-autonomous thymocyte developmental block associated with apoptosis at the ß-selection stage, independent of T cell receptor ß recombination. No abnormalities are observed in the B cell lineage. Transgenes encoding wild-type or DNA-binding-deficient mutant Aif rectify the thymic defect, but a transgene encoding oxidoreductase activity-deficient mutant Aif does not. The Hq thymic block is reversed in vivo by antioxidant treatment, and Hq T but not B lineage cells show enhanced oxidative stress. Thus, Aif, a ubiquitous protein, serves a lineage-specific nonredundant antiapoptotic role in the T cell lineage by regulating reactive oxygen species during thymic ß-selection.
Assuntos
Fator de Indução de Apoptose/fisiologia , Linfócitos T/fisiologia , Animais , Apoptose , Morte Celular , Linhagem da Célula , DNA/metabolismo , Feminino , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Espécies Reativas de Oxigênio/metabolismo , Recombinação Genética , Timócitos/metabolismo , Timócitos/patologia , Timo/crescimento & desenvolvimento , Timo/patologiaRESUMO
Poor T cell immunity is one of the many defects seen in elderly humans and aged (Ad) mice. We report that naive CD4 T cells from aged mice (ANCD4 cells) showed greater apoptosis upon primary activation than those from young (Yg) mice, with loss of mitochondrial membrane potential, poor activation of Rel family transcription factors and increased DNA damage. Their ability to enhance glycolysis, produce lactate and induce autophagy following activation was also compromised. ANCD4 cells remained susceptible to death beyond first cell division. Activated ANCD4 cells also showed poor transition to a 'central memory' (CM) CD44(high), CD62L(high) phenotype in vitro. This correlated with low proportions of CM cells in Ad mice in vivo. Functionally, too, IFN-gamma responses recalled from T cells of immunized Ad mice, poor to begin with, worsened with time as compared with Yg mice. Thus, ANCD4 cells handle activation-associated stress very poorly due to multiple defects, possibly contributing to poor formation of long-lasting memory.
Assuntos
Envelhecimento/imunologia , Apoptose/imunologia , Linfócitos T CD4-Positivos/imunologia , Reparo do DNA/imunologia , Ativação Linfocitária , NF-kappa B/imunologia , Fatores Etários , Animais , Anticorpos Monoclonais/imunologia , Apoptose/efeitos dos fármacos , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Ovalbumina/imunologiaRESUMO
Cellular and molecular mechanisms involved in aging are notoriously complex. Aging-related immune decline of T lymphocyte function is partly caused by attrition of thymic T cell development, which involves programmed creation and repair of DNA breaks for generating T cell receptors. Aging also leads to significant alterations in the cellular DNA repair ability. We show that higher levels of gamma-phosphorylated H2AX (pH2AX), which marks DNA double-stranded breaks (DSBs), were detectable in early thymocyte subsets of aged as compared to young mice. Also, while only 1-2 foci of nuclear accumulation of pH2AX were detectable in these early thymocytes from young mice, cells from aged mice showed higher numbers of pH2AX foci. In CD4-CD8- double-negative (DN) thymocytes of aged mice, which showed the highest levels of DSBs, there was a modest increase in levels of the DNA repair protein MRE11, but not of either Ku70, another DNA repair protein, or the cell cycle checkpoint protein p53. Thus, immature thymocytes in aged mice show a marked increase in DNA DSBs with only a modest enhancement of repair processes, and the resultant cell cycle block could contribute to aging-related defects of T cell development.
Assuntos
Envelhecimento/imunologia , Antígenos de Diferenciação de Linfócitos T/biossíntese , Quebras de DNA de Cadeia Dupla , Interleucina-2/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/metabolismo , Envelhecimento/genética , Animais , Separação Celular , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Interleucina-2/genética , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Apoptosis-inducing factor (Aif) is a mitochondrial flavoprotein with multiple roles in apoptosis as well as in cellular respiration and redox regulation. The harlequin (Hq) mouse strain carries an aif locus modification causing reduced Aif expression. We demonstrate that activated CD4(+) and CD8(+) peripheral T cells from Hq mice show resistance to neglect-induced death (NID) triggered by growth factor withdrawal, but not to death induced by multiple agents that trigger DNA damage. Aif translocates to the nucleus in cells undergoing NID, and, in Hq T cell blasts, resistance to NID is associated with reduced cytosolic release of mitochondrial cytochrome c, implicating Aif in this event. In contrast, Hq T cell blasts express higher levels of CD95L, demonstrating increased susceptibility to activation-induced cell death (AICD) and apoptosis triggered by hydrogen peroxide. Superoxide scavenging protects from AICD in wild-type, but not Hq, T cell blasts, suggesting that Aif plays a crucial superoxide-scavenging role to regulate T cell AICD. Finally, the altered pattern of death susceptibility is reproduced by siRNA-mediated reduction of Aif expression in normal T cells. Thus, Aif serves nonredundant roles, both proapoptotic and antiapoptotic, in activated peripheral T cells.
Assuntos
Fator de Indução de Apoptose/metabolismo , Apoptose/fisiologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Animais , Fator de Indução de Apoptose/imunologia , Western Blotting , Diferenciação Celular/imunologia , Citometria de Fluxo , Camundongos , Camundongos Mutantes , Microscopia Confocal , Mitocôndrias/metabolismo , Mitocôndrias/patologia , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/citologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The progeny of T lymphocytes responding to immunization mostly die rapidly, leaving a few long-lived survivors functioning as immune memory. Thus, control of this choice of death versus survival is critical for immune memory. There are indications that reactive radicals may be involved in this death pathway. We now show that, in mice lacking inducible nitric oxide synthase (iNOS), higher frequencies of both CD4 and CD8 memory T cells persist in response to immunization, even when iNOS(+/+) APCs are used for immunization. Postactivation T cell death by neglect is reduced in iNOS(-/-) T cells, and levels of the antiapoptotic proteins Bcl-2 and Bcl-xL are increased. Inhibitors of the iNOS-peroxynitrite pathway also enhance memory responses and block postactivation death by neglect in both mouse and human T cells. However, early primary immune responses are not enhanced, which suggests that altered survival, rather than enhanced activation, is responsible for the persistent immunity observed. Thus, in primary immune responses, iNOS in activated T cells autocrinely controls their susceptibility to death by neglect to determine the level of persisting CD4 and CD8 T cell memory, and modulation of this pathway can enhance the persistence of immune memory in response to vaccination.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD8-Positivos/enzimologia , Morte Celular/fisiologia , Memória Imunológica/fisiologia , Óxido Nítrico Sintase/metabolismo , Clorometilcetonas de Aminoácidos/metabolismo , Animais , Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Ácido Peroxinitroso/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Baço/citologia , Subpopulações de Linfócitos T , Proteína bcl-XRESUMO
Memory B cells of mice with Ig mu transgenes often carry transgene copies that have moved into the Igh locus via somatic translocation. This phenomenon has been attributed to a selection pressure for somatic hypermutations, which generally are observed at much higher frequencies in translocated copies than in ectopic copies. We tested this idea by immunizing Ig-mu transgenic mice in a manner designed to select B cells that required only one V(H) mutation for a switch in antigenic specificity and recruitment into the memory pool. Despite the minimal mutation requirement, hybridomas carrying somatic translocations to the Igh locus were obtained. Importantly, this occurred despite the fact that translocated and untranslocated mu-transgenes were mutated comparably. Evidently, a strong selection advantage was conferred upon B cells by the somatic translocations. Among the hybridomas, translocated mu-transgenes were active, while ectopic mu-transgenes were uniformly silent. The translocated copy that had conferred an affinity-based selection advantage was expressed at the highest level. Moreover, translocated copies were differentially expressed among hybridoma members, which belonged to a common post-mutational lineage. This suggests that adjustments in transgene expression levels had occurred during memory cell development. These results indicate that, apart from their potential influences on somatic hypermutagenesis and class switch recombination, elements in the Igh locus promote the selection of memory B cells in another way, possibly by regulating the level of Ig expression at various stages of antigen-driven differentiation.
Assuntos
Subpopulações de Linfócitos B/imunologia , Regulação da Expressão Gênica/imunologia , Genes de Imunoglobulinas , Cadeias mu de Imunoglobulina/genética , Memória Imunológica/genética , Transgenes , Animais , Ácido Arsanílico/imunologia , Sequência de Bases , Diferenciação Celular , Linhagem da Célula , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Hemocianinas/imunologia , Hibridomas/imunologia , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/genética , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologiaRESUMO
Modalities for inducing long-lasting immune responses are essential components of vaccine design. Most currently available immunological adjuvants empirically used for this purpose cause some inflammation, limiting clinical acceptability. We show that pentoxifylline (PF), a phosphodiesterase (PDE) inhibitor in common clinical use, enhances long-term persistence of T cell responses, including protective responses to a bacterial immunogen, Salmonella typhimurium, via a cAMP-dependent protein kinase A-mediated effect on T cells if given to mice for a brief period during immunization. PF inhibits activation-mediated loss of superantigen-reactive CD4 as well as CD8 T cells in vivo without significantly affecting their activation, and inhibits activation-induced death and caspase induction in stimulated CD4 as well as CD8 T cells in vitro without preventing the induction of activation markers. Consistent with this ability to prevent activation-induced death in not only CD4 but also CD8 T cells, PF also enhances the persistence of CD8 T cell responses in vivo. Thus, specific inhibition of activation-induced T cell apoptosis transiently during immune priming is likely to enhance the persistence of CD4 and CD8 T cell responses to vaccination, and pharmacological modulators of the cAMP pathway already in clinical use can be used for this purpose as immunological adjuvants.