RESUMO
Dendritic cells (DCs) are the most potent APCs for activating naive T cells, a process facilitated by the ability of immature DCs to mature and home to lymph nodes after encountering an inflammatory stimulus. Proteins involved in cytoskeletal rearrangement play an important role in regulating the adherence and motility of DCs. Vav1, a guanine nucleotide exchange factor for Rho family GTPases, mediates cytoskeletal rearrangement in hematopoietic cells following integrin ligation. We show that Vav1 is not required for the normal maturation of DCs in vitro; however, it is critical for DC binding to fibronectin and regulates the distribution but not the formation of podosomes. We also found that DC Vav1 was an important component of a signaling pathway involving focal adhesion kinase, phospholipase C-gamma2, and ERK1/2 following integrin ligation. Surprisingly, Vav1(-/-) DCs had increased rates of migration in vivo compared with wild-type control DCs. In vitro findings show that the presence of adhesive substrates such as fibronectin resulted in inhibition of migration. However, there was less inhibition in the absence of Vav1. These findings suggest that DC migration is negatively regulated by adhesion and integrin-mediated signaling and that Vav1 has a central role in this process.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Proteínas Proto-Oncogênicas c-vav/fisiologia , Animais , Adesão Celular/genética , Adesão Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Movimento Celular/genética , Células Cultivadas , Células Dendríticas/metabolismo , Fibronectinas/metabolismo , Integrinas/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/genética , Ligação Proteica/imunologia , Proteínas Proto-Oncogênicas c-vav/biossíntese , Proteínas Proto-Oncogênicas c-vav/deficiência , Proteínas Proto-Oncogênicas c-vav/genética , Pseudópodes/genética , Pseudópodes/imunologia , Pseudópodes/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Within the past decade, the critical roles of T cells and T cell-mediated immunity in inflammation-induced osteoclastogenesis and subsequent bone loss have been extensively studied, thereby establishing the new paradigm of osteoimmunology. Therefore, dendritic cells (DCs), the most potent antigen-presenting cells, responsible for activation of naïve T cells and orchestration of the immune response, became critically situated at the osteo-immune interface. Today, emerging new evidence suggests that DC may be directly involved in inflammation-induced osteoclastogenesis and bone loss, by acting as osteoclast (OC) precursors that can further develop into DC-derived OCs (DDOC) under inflammatory conditions. These findings have tremendous implications, because in addition to DC's important roles in regulating innate and adaptive immunity, a direct contribution by these cells to inflammation-induced bone loss may provide a promising therapeutic target not only for controlling inflammation but also for modulating bone destruction.
Assuntos
Reabsorção Óssea/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Inflamação/complicações , Animais , Doenças Ósseas/complicações , Doenças Ósseas/etiologia , Doenças Ósseas/imunologia , Reabsorção Óssea/etiologia , Linhagem da Célula/imunologia , Células Dendríticas/citologia , Humanos , Inflamação/imunologia , Modelos Imunológicos , Osteoclastos/citologia , Osteoclastos/imunologiaRESUMO
Diabetic patients experience a higher risk for severe periodontitis; however, the underlying mechanism remains unclear. We investigated the contribution of antibacterial T-cell-mediated immunity to enhanced alveolar bone loss during periodontal infection in nonobese diabetic (NOD) mice by oral inoculation with Actinobacillus actinomycetemcomitans, a G(-) anaerobe responsible for juvenile and severe periodontitis. The results show that 1) inoculation with A. actinomycetemcomitans in pre-diabetic NOD mice does not alter the onset, incidence, and severity of diabetes; 2) after A. actinomycetemcomitans inoculation, diabetic NOD mice (blood glucose >200 mg/dl and with severe insulitis) exhibit significantly higher alveolar bone loss compared with pre-diabetic and nondiabetic NOD mice; and 3) A. actinomycetemcomitans-reactive CD4+ T-cells in diabetic mice exhibit significantly higher proliferation and receptor activator of nuclear factor kappaB ligand (RANKL) expression. When diabetic mice are treated with the RANKL antagonist osteoprotegerin (OPG), there is a significant reversal of alveolar bone loss, as well as reduced RANKL expression in A. actinomycetemcomitans-reactive CD4+ T-cells. This study clearly describes the impact of autoimmunity to anaerobic infection in an experimental periodontitis model of type 1 diabetes. Thus, microorganism-reactive CD4+ T-cells and the RANKL-OPG axis provide the molecular basis of the advanced periodontal breakdown in diabetes and, therefore, OPG may hold therapeutic potential for treating bone loss in diabetic subjects at high risk.
Assuntos
Infecções por Actinobacillus/imunologia , Aggregatibacter actinomycetemcomitans , Perda do Osso Alveolar/fisiopatologia , Linfócitos T CD4-Positivos/imunologia , Proteínas de Transporte/fisiologia , Diabetes Mellitus Tipo 1/microbiologia , Glicoproteínas de Membrana/fisiologia , Perda do Osso Alveolar/microbiologia , Animais , Bactérias Anaeróbias , Linfócitos T CD4-Positivos/microbiologia , Diabetes Mellitus Tipo 1/imunologia , Glicoproteínas/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Osteoprotegerina , Estado Pré-Diabético/imunologia , Estado Pré-Diabético/microbiologia , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologiaRESUMO
The renin-angiotensin system (RAS) is related to physiological adaptations induced by exercise. Angiotensin-converting enzyme (ACE) 2 is a major regulator of the RAS in tissues, as it metabolizes angiotensin (Ang) II to Ang-(1-7). The aim of this study was to determine the effects of ACE2 deficiency on physical performance and physiological adaptations induced by voluntary running. Physical performance, body composition and plasma angiotensin levels, as well as tissue morphology and gene expression of RAS components in the left ventricle (LV) and skeletal muscle (gastrocnemius), were evaluated in ACE2-deficient (ACE2(-/y)) and wild-type (ACE2(+/y)) mice after 6 weeks of voluntary wheel running. ACE2(-/y) mice run less than ACE2(+/y) mice (19±4.7 vs. 26±12.6 revolutions per day × 100, P<0.01). The ACE2(+/y) group presented a lower fat mass (15±1.1%) and higher muscle mass (76.6±1.6%) after 6 weeks of voluntary running compared with the sedentary control group (fat mass: 18.3±2.1%; muscle mass: 72.7±2.2). However, no change in body composition was observed in ACE2(-/y) mice after exercise. Heart and skeletal muscle hypertrophy was observed only in trained ACE2(+/y) mice. Besides a small decrease in Ang I in ACE2(-/y) mice, plasma levels of angiotensin peptides remained unchanged by exercise or ACE2 deficiency. In the LV of trained animals, AT2 gene expression was higher in ACE2(+/y) compared with ACE2(-/y) mice. ACE2 deficiency leads to an increase in AT1 gene expression in skeletal muscle. ACE expression in soleus was increased in all exercised groups. ACE2 deficiency affects physical performance and impairs cardiac and skeletal muscle adaptations to exercise.
Assuntos
Adaptação Fisiológica , Coração/fisiologia , Músculo Esquelético/fisiologia , Peptidil Dipeptidase A/deficiência , Enzima de Conversão de Angiotensina 2 , Angiotensinas/sangue , Animais , Exercício Físico , Teste de Esforço , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MiocárdioRESUMO
Src homology domain 2-containing inositol 5-phosphatases 1 and 2 (SHIP1 and SHIP2) are capable of dephosphorylating the second messenger PtdIns(3,4,5) P3 (phosphatidylinositol 3,4,5-trisphosphate) and interacting with several signalling proteins. SHIP1 is essentially expressed in haematopoietic cells, whereas SHIP2, a closely related enzyme, is ubiquitous. In the present study, we show that SHIP1 and SHIP2 are expressed as functional PtdIns(3,4,5) P3 5-phosphatases in human blood platelets and are capable of interacting when these two lipid phosphatases are co-expressed, either naturally (platelets and A20 B lymphoma cells) or artificially (COS-7 cells). Using COS-7 cells transfected with deletion mutants of SHIP2, we demonstrate that the Src homology domain 2 of SHIP2 is the minimal and sufficient protein motif responsible for the interaction between the two phosphatases. These results prompted us to investigate the relative importance of SHIP1 and SHIP2 in the control of PtdIns(3,4,5) P3 levels in platelets using homozygous or heterozygous SHIP1- or SHIP2-deficient mice. Our results strongly suggest that SHIP1, rather than SHIP2, plays a major role in controlling PtdIns(3,4,5) P3 levels in response to thrombin or collagen activation of mouse blood platelets.
Assuntos
Plaquetas/enzimologia , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Animais , Plaquetas/efeitos dos fármacos , Células COS , Linhagem Celular Tumoral , Citoesqueleto/enzimologia , Humanos , Inositol Polifosfato 5-Fosfatases , Camundongos , Camundongos Knockout , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/química , Fosforilação , Trombina/farmacologia , Tirosina/metabolismo , Domínios de Homologia de srcRESUMO
BACKGROUND AND OBJECTIVES: Angiotensin-converting enzyme 2 (ACE2) converts angiotensin II (Ang1-8) to angiotensin 1-7 (Ang1-7), a functional antagonist of Ang1-8, with vasodilatory, antiproliferative, antiangiogenic, and anti-inflammatory properties. In conditions with an unbalanced renin-angiotensin-aldosterone system with elevated Ang1-8, administration of ACE2 has shown promising effects in a variety of animal models. Enhancing ACE2 activity by exogenous administration of ACE2 might also be beneficial in human diseases with pathologically elevated Ang1-8. As a first step we performed a first-in-man study to determine pharmacokinetics, pharmacodynamics, safety, and tolerability of recombinant ACE2 in healthy volunteers. METHODS: Recombinant human ACE2 (rhACE2) was administered intravenously to healthy human subjects in a randomized, double-blind, placebo-controlled, single-dose, dose-escalation study followed by an open-label multiple-dose study. ACE2 concentrations were determined by quantifying ACE2 activity and ACE2 content in plasma samples. Concentrations of the angiotensin system effector peptides Ang1-8, Ang1-7, and Ang1-5 were determined using a liquid chromatography-tandem mass spectrometry method. RESULTS: Single rhACE2 doses of 100-1,200 µg/kg caused a dose-dependent increase of systemic exposure with biphasic elimination and a dose-independent terminal half-life of 10 h. In all single-dose cohorts, Ang1-8 decreased within 30 min postinfusion, angiotensin 1-7 (Ang1-7) either increased (100 and 200 µg/kg doses), decreased, or remained unchanged (400-1,200 µg/kg doses), whereas angiotensin 1-5 (Ang1-5) transiently increased for all doses investigated. With the exception of the lowest rhACE2 dose, the decrease in Ang1-8 levels lasted for at least 24 h. Repeated dosing (400 µg/kg for 3 or 6 days) caused only minimal accumulation of ACE2, and Ang1-8 levels were suppressed over the whole application period. CONCLUSIONS: Administration of rhACE2 was well tolerated by healthy human subjects. Exposure was dose dependent with a dose-independent terminal elimination half-life in the range of 10 h. Despite marked changes in angiotensin system peptide concentrations, cardiovascular effects were absent, suggesting the presence of effective compensatory mechanisms in healthy volunteers.
Assuntos
Peptidil Dipeptidase A/farmacocinética , Proteínas Recombinantes/farmacocinética , Angiotensina I/sangue , Angiotensina II/sangue , Enzima de Conversão de Angiotensina 2 , Método Duplo-Cego , Feminino , Humanos , Masculino , Fragmentos de Peptídeos/sangue , Peptidil Dipeptidase A/administração & dosagem , Proteínas Recombinantes/administração & dosagemRESUMO
Despite the established role for PI3Ks in cell migration, the PI3Ks involved in lymphocyte chemotaxis are poorly defined. In this study, we report that p110gamma-deficient T cells, but not B cells, show reduced chemotactic responses to the lymphoid chemokines, CCL19, CCL21, and CXCL12. As B cell and T cell chemotactic responses were both sensitive to the general PI3K inhibitors, wortmannin (WMN) and LY294002, we explored whether B cell responses were affected in mice lacking p110delta, a major PI3K isoform in lymphocytes. B cells deficient in p110delta showed diminished chemotactic responses, especially to CXCL13. Adoptive transfer experiments with WMN-treated wild-type B cells and with p110delta-deficient B cells revealed diminished homing to Peyer's patches and splenic white pulp cords. WMN selectively inhibited CXCR5-dependent B cell homing to Peyer's patches. These observations establish that p110gamma and p110delta function in lymphocyte chemotaxis, and show differential roles for PI3K family members in B and T cell migration.