RESUMO
Phagocytosis is a specialized process that enables cellular ingestion and clearance of microbes, dead cells and tissue debris that are too large for other endocytic routes. As such, it is an essential component of tissue homeostasis and the innate immune response, and also provides a link to the adaptive immune response. However, ingestion of large particulate materials represents a monumental task for phagocytic cells. It requires profound reorganization of the cell morphology around the target in a controlled manner, which is limited by biophysical constraints. Experimental and theoretical studies have identified critical aspects associated with the interconnected biophysical properties of the receptors, the membrane, and the actin cytoskeleton that can determine the success of large particle internalization. In this review, we will discuss the major physical constraints involved in the formation of a phagosome. Focusing on two of the most-studied types of phagocytic receptors, the Fcγ receptors and the complement receptor 3 (αMß2 integrin), we will describe the complex molecular mechanisms employed by phagocytes to overcome these physical constraints.
Assuntos
Fagocitose/imunologia , Fagocitose/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Fenômenos Biofísicos , Movimento Celular/imunologia , Movimento Celular/fisiologia , Extensões da Superfície Celular/imunologia , Extensões da Superfície Celular/fisiologia , Humanos , Ligantes , Antígeno de Macrófago 1/química , Antígeno de Macrófago 1/imunologia , Antígeno de Macrófago 1/fisiologia , Modelos Imunológicos , Miosina Tipo II/imunologia , Miosina Tipo II/fisiologia , Fagossomos/imunologia , Fagossomos/fisiologia , Conformação Proteica , Pseudópodes/imunologia , Pseudópodes/fisiologia , Receptores de IgG/química , Receptores de IgG/imunologia , Receptores de IgG/fisiologiaRESUMO
Activation of naive T cells by antigen-presenting cells (APCs) is an essential step in mounting an adaptive immune response. It is known that antigen recognition and T cell receptor (TCR) signaling depend on forces applied by the T cell actin cytoskeleton, but until recently, the underlying mechanisms have been poorly defined. Here, we review recent advances in the field, which show that specific actin-dependent structures contribute to the process in distinct ways. In essence, T cell priming involves a tug-of-war between the cytoskeletons of the T cell and the APC, where the actin cytoskeleton serves as a mechanical intermediate that integrates force-dependent signals. We consider each of the relevant actin-rich T cell structures separately and address how they work together at the topologically and temporally complex cell-cell interface. In addition, we address how this mechanobiology can be incorporated into canonical immunological models to improve how these models explain T cell sensitivity and antigenic specificity.
Assuntos
Citoesqueleto de Actina/genética , Actinas/genética , Células Apresentadoras de Antígenos/imunologia , Sinapses Imunológicas/genética , Mecanotransdução Celular , Citoesqueleto de Actina/imunologia , Actinas/imunologia , Imunidade Adaptativa/imunologia , Comunicação Celular/imunologia , Citoesqueleto/genética , Citoesqueleto/imunologia , Humanos , Sinapses Imunológicas/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Modelos Imunológicos , Pseudópodes/imunologia , Pseudópodes/ultraestrutura , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/genética , Linfócitos T/imunologiaRESUMO
The ability of phagocytes to recognize, immobilize, and engulf extracellular targets are fundamental immune cell processes that allow for the destruction of a variety of microbial intruders. The phagocytic process depends onsignalling events that initiate dynamic changes in the plasma membrane architecture that are required to accommodate the internalization of large particulate targets. To better understand fundamental molecular mechanisms responsible for facilitating phagocytic receptor-mediated regulation of cytoskeletal networks, our research has focused on investigating representative immunoregulatory proteins from the channel catfish (Ictalurus punctatus) leukocyte immune-type receptor family (IpLITRs). Specifically, we have shown that a specific IpLITR-type can regulate the constitutive deployment of filopodial-like structures to actively capture and secure targets to the phagocyte surface, which is followed by F-actin mediated membrane dynamics that are associated with the formation of phagocytic cup-like structures that precede target engulfment. In the present study, we use confocal imaging to examine the recruitment of mediators of the F-actin cytoskeleton during IpLITR-mediated regulation of membrane dynamics. Our results provide novel details regarding the dynamic recruitment of the signaling effectors Nck and Syk during classical as well as atypical IpLITR-induced phagocytic processes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Ictaluridae/imunologia , Proteínas Oncogênicas/imunologia , Fagocitose/imunologia , Receptores Imunológicos/imunologia , Quinase Syk/imunologia , Animais , Linhagem Celular , Fibroblastos , Pseudópodes/imunologia , RatosRESUMO
Neutrophil chemotaxis is essential in responses to infection and underlies inflammation. In neutrophils, the small GTPase Rac1 has discrete functions at both the leading edge and in the retraction of the trailing structure at the cell's rear (uropod), but how Rac1 is regulated at the uropod is unknown. Here, we identified a mechanism mediated by the trafficking protein synaptotagmin-like 1 (SYTL1 or JFC1) that controls Rac1-GTP recycling from the uropod and promotes directional migration of neutrophils. JFC1-null neutrophils displayed defective polarization and impaired directional migration to N-formyl-methionine-leucyl-phenylalanine in vitro, but chemoattractant-induced actin remodeling, calcium signaling and Erk activation were normal in these cells. Defective chemotaxis was not explained by impaired azurophilic granule exocytosis associated with JFC1 deficiency. Mechanistically, we show that active Rac1 localizes at dynamic vesicles where endogenous JFC1 colocalizes with Rac1-GTP. Super-resolution microscopy (STORM) analysis shows adjacent distribution of JFC1 and Rac1-GTP, which increases upon activation. JFC1 interacts with Rac1-GTP in a Rab27a-independent manner to regulate Rac1-GTP trafficking. JFC1-null cells exhibited Rac1-GTP accumulation at the uropod and increased tail length, and Rac1-GTP uropod accumulation was recapitulated by inhibition of ROCK or by interference with microtubule remodeling. In vivo, neutrophil dynamic studies in mixed bone marrow chimeric mice show that JFC1-/- neutrophils are unable to move directionally toward the source of the chemoattractant, supporting the notion that JFC1 deficiency results in defective neutrophil migration. Our results suggest that defective Rac1-GTP recycling from the uropod affects directionality and highlight JFC1-mediated Rac1 trafficking as a potential target to regulate chemotaxis in inflammation and immunity.
Assuntos
Quimiotaxia/imunologia , Guanosina Trifosfato/imunologia , Proteínas de Membrana/imunologia , Neuropeptídeos/imunologia , Neutrófilos/imunologia , Pseudópodes/imunologia , Proteínas de Transporte Vesicular/imunologia , Proteínas rac1 de Ligação ao GTP/imunologia , Animais , Quimiotaxia/genética , Guanosina Trifosfato/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Neutrófilos/patologia , Pseudópodes/genética , Pseudópodes/patologia , Proteínas de Transporte Vesicular/genética , Proteínas rab27 de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP/imunologia , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Neutrophils are the first leukocytes to arrive at sites of injury during the acute inflammatory response. To maintain the polarized morphology during migration, nonmuscle myosins class II are essential, but studies using genetic models to investigate the role of Myh9 for neutrophil migration were missing. In this study, we analyzed the functional role of Myh9 on neutrophil trafficking using genetic downregulation of Myh9 in Vav-iCre+/Myh9wt/fl mice because the complete knockout of Myh9 in the hematopoietic system was lethal. Migration velocity and Euclidean distance were significantly diminished during mechanotactic migration of Vav-iCre+/Myh9wt/fl neutrophils compared with Vav-iCre-/Myh9wt/fl control neutrophils. Similar results were obtained for transmigration and migration in confined three-dimensional environments. Stimulated emission depletion nanoscopy revealed that a certain threshold of Myh9 was required to maintain proper F-actin dynamics in the front of the migrating cell. In laser-induced skin injury and in acute peritonitis, reduced Myh9 expression in the hematopoietic system resulted in significantly diminished neutrophil extravasation. Investigation of bone marrow chimeric mice in the peritonitis model revealed that the migration defect was cell intrinsic. Expression of Myh9-EGFP rescued the Myh9-related defects in two-dimensional and three-dimensional migration of Hoxb8-SCF cell-derived neutrophils generated from fetal liver cells with a Myh9 knockdown. Live cell imaging provided evidence that Myh9 was localized in branching lamellipodia and in the uropod where it may enable fast neutrophil migration. In summary, the severe migration defects indicate an essential and fundamental role of Myh9 for neutrophil trafficking in innate immunity.
Assuntos
Movimento Celular/imunologia , Imunidade Inata , Infiltração de Neutrófilos , Neutrófilos/imunologia , Miosina não Muscular Tipo IIA/imunologia , Pseudópodes/imunologia , Actinas/genética , Actinas/imunologia , Animais , Movimento Celular/genética , Camundongos , Camundongos Transgênicos , Cadeias Pesadas de Miosina , Neutrófilos/patologia , Miosina não Muscular Tipo IIA/genética , Peritonite/genética , Peritonite/imunologia , Peritonite/patologia , Pseudópodes/genética , Pele/imunologia , Pele/lesões , Pele/patologiaRESUMO
Phagocytosis evolved from a fundamental nutrient acquisition mechanism in primitive unicellular amoeboids, into a dynamic and complex component of innate immunity in multicellular organisms. To better understand the cellular mechanisms contributing to phagocytic processes across vertebrates, our research has focused on characterizing the involvement of innate immune proteins originally identified in channel catfish (Ictalurus punctatus) called leukocyte immune-type receptors (IpLITRs). These unique teleost proteins share basic structural as well as distant phylogenetic relationships with several immunoregulatory proteins within the mammalian immunoglobulin superfamily. In the present study, we use a combination of live-cell confocal imaging and high-resolution scanning electron microscopy to further examine the classical immunoreceptor tyrosine-based activation motif (ITAM)-dependent phagocytic pathway mediated by the chimeric construct IpLITR 2.6b/IpFcRγ-L and the functionally diverse immunoreceptor tyrosine-based inhibitory motif-containing receptor IpLITR 1.1b. Results demonstrate that IpLITR 1.1b-expressing cells can uniquely generate actin-dense filopodia-like protrusions during the early stages of extracellular target interactions. In addition, we observed that these structures retract after contacting extracellular targets to secure captured microspheres on the cell surface. This activity was often followed by the generation of robust secondary waves of actin polymerization leading to the formation of stabilized phagocytic cups. At depressed temperatures of 27°C, IpLITR 2.6b/IpFcRγ-L-mediated phagocytosis was completely blocked, whereas IpLITR 1.1b-expressing cells continued to generate dynamic actin-dense filopodia at this lower temperature. Overall, these results provide new support for the hypothesis that IpLITR 1.1b, but not IpLITR 2.6b/IpFcRγ-L, directly triggers filopodia formation when expressed in representative myeloid cells. This also offers new information regarding the directed ability of immunoregulatory receptor-types to initiate dynamic membrane structures and provides insights into an alternative ITAM-independent target capture pathway that is functionally distinct from the classical phagocytic pathways.
Assuntos
Citoesqueleto/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Fagócitos/imunologia , Fagócitos/metabolismo , Pseudópodes/imunologia , Pseudópodes/metabolismo , Receptores Imunológicos/metabolismo , Animais , Peixes , Expressão Gênica , Leucócitos/ultraestrutura , Fagocitose/genética , Fagocitose/imunologia , Ligação Proteica , Receptores Fc/metabolismo , Receptores Imunológicos/genética , TemperaturaRESUMO
Global bursts in free intracellular calcium (Ca2+) are among the most conspicuous signaling events in immune cells. To test the common view that Ca2+ bursts mediate rearrangement of the actin cytoskeleton in response to the activation of G protein-coupled receptors, we combined single-cell manipulation with fluorescence imaging and monitored the Ca2+ concentration in individual human neutrophils during complement-mediated chemotaxis. By decoupling purely chemotactic pseudopod formation from cell-substrate adhesion, we showed that physiological concentrations of anaphylatoxins, such as C5a, induced nonadherent human neutrophils to form chemotactic pseudopods but did not elicit Ca2+ bursts. By contrast, pathological or supraphysiological concentrations of C5a often triggered Ca2+ bursts, but pseudopod protrusion stalled or reversed in such cases, effectively halting chemotaxis, similar to sepsis-associated neutrophil paralysis. The maximum increase in cell surface area during pseudopod extension in pure chemotaxis was much smaller-by a factor of 8-than the known capacity of adherent human neutrophils to expand their surface. Because the measured rise in cortical tension was not sufficient to account for this difference, we attribute the limited deformability to a reduced ability of the cytoskeleton to generate protrusive force in the absence of cell adhesion. Thus, we hypothesize that Ca2+ bursts in neutrophils control a mechanistic switch between two distinct modes of cytoskeletal organization and dynamics. A key element of this switch appears to be the expedient coordination of adhesion-dependent lock or release events of cytoskeletal membrane anchors.
Assuntos
Cálcio/imunologia , Quimiotaxia/imunologia , Neutrófilos/imunologia , Pseudópodes/imunologia , Citoesqueleto de Actina/imunologia , Citoesqueleto de Actina/metabolismo , Cálcio/metabolismo , Adesão Celular/imunologia , Complemento C5a/imunologia , Complemento C5a/metabolismo , Humanos , Microscopia de Fluorescência , Neutrófilos/citologia , Neutrófilos/metabolismo , Pseudópodes/metabolismo , Receptor da Anafilatoxina C5a/imunologia , Receptor da Anafilatoxina C5a/metabolismo , Transdução de Sinais/imunologia , Análise de Célula Única/métodosRESUMO
Gelatin, denatured collagen, temporarily exists in tissues and may well be pathophysiologically involved in tissue remodeling, inflammation or tissue damage. The present study is aimed to investigate possible biological roles of gelatin by examining its effects on monocyte-like histiocytic lymphoma cell line U937. Once stimulated by phorbol 12-myristate 13-acetate (PMA), U937 cells differentiate into macrophage-like cells, changing from non-adherent to adherent cells with extended pseudopodia. Here we pre-treated the cell dishes with gelatin solution for cell culture. Interestingly, we found that PMA-stimulated U937 cells formed multicellular aggregates on gelatin-coated dishes, accompanying NF-κB-mediated production of pro-inflammatory cytokines, whereas cell aggregation was not detected on non-coated dishes. Moreover, differentiated U937 cells on gelatin-coated dishes showed increased autophagy level and endocytosis. Surprisingly, formation of multicellular aggregates and pro-inflammatory cytokine production were both attenuated by either down-regulation of autophagy with inhibitors, such as 3-methyladenine (3MA) or chloroquine (CQ), or repression of endocytosis with siRNA targeting Endo180. Moreover, autophagy was inhibited by si-Endo180, and endocytosis was suppressed by 3MA, suggesting a positive feedback loop between autophagy and endocytosis. The results revealed that gelatin-coating induced differentiated U937 cells to form cell aggregates and promote NF-κB-mediated pro-inflammatory cytokine production at least partially through an endocytosis-autophagy pathway.
Assuntos
Autofagia/efeitos dos fármacos , Citocinas/metabolismo , Endocitose/efeitos dos fármacos , Matriz Extracelular/metabolismo , Gelatina/metabolismo , Macrófagos/efeitos dos fármacos , Acetato de Tetradecanoilforbol/análogos & derivados , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Proteína Beclina-1/antagonistas & inibidores , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Carcinógenos/farmacologia , Agregação Celular , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cloroquina/farmacologia , Gelatina/isolamento & purificação , Humanos , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Lectinas de Ligação a Manose/antagonistas & inibidores , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/imunologia , Pseudópodes/metabolismo , Interferência de RNA , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Pele/química , Sus scrofa , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells.
Assuntos
Interações Hospedeiro-Patógeno/genética , MicroRNAs/genética , Shigella/genética , Shigella/virologia , Replicação Viral/genética , Linhagem Celular , Replicação do DNA/genética , Perfilação da Expressão Gênica/métodos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Pseudópodes/imunologia , Interferência de RNA/fisiologiaRESUMO
Macrophages have multiple functions in both inhibiting and promoting hepatocarcinogenesis, which are dependent on their phenotypes. Thus, we were interested in clarifying the 'training' proinflammatory effects exerted by barley ß-glucan (BBG) on monocyte-derived macrophages from patients with hepatocellular carcinoma (HCC-Mfs). After isolation and differentiation HCC-Mfs were treated with different concentrations of BBG and functional assays were then conducted after 24 h, and 3 and 5 days of incubation. The release of reactive oxygen species, arginase concentration and cell morphology were analyzed. Under the influence of BBG neoplastic cells slightly elongated and dendric-like filopodia were observed. In HCC-Mfs, the significant generation of NO and O2- was seen on days 3 and 5 of culture, concomitantly with significant depletion (p<0.05) of arginase activity. In summary, we showed that HCC-Mfs, 'trained' in a BBG microenvironment keep a highly dynamic plasticity, together with their proinflammatory polarization, expressed by reactive oxygen species and reactive nitrogen intermediates (RNI) augmentation.
Assuntos
Microambiente Celular/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , beta-Glucanas/imunologia , Animais , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Células Cultivadas , Microambiente Celular/efeitos dos fármacos , Feminino , Hordeum/química , Humanos , Inflamação/imunologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Óxido Nítrico/imunologia , Óxido Nítrico/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/imunologia , Ratos Wistar , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Especificidade da Espécie , beta-Glucanas/farmacologiaRESUMO
Exploring the mechanisms controlling lymphocyte trafficking is essential for understanding the function of the immune system and the pathophysiology of immunodeficiencies. The mammalian Ste20-like kinase 1 (Mst1) has been identified as a critical signaling mediator of T cell migration, and loss of Mst1 results in immunodeficiency disease. Although Mst1 is known to support T cell migration through induction of cell polarization and lamellipodial formation, the downstream effectors of Mst1 are incompletely defined. Mice deficient for the actin-bundling protein L-plastin (LPL) have phenotypes similar to mice lacking Mst1, including decreased T cell polarization, lamellipodial formation, and cell migration. We therefore asked whether LPL functions downstream of Mst1. The regulatory N-terminal domain of LPL contains a consensus Mst1 phosphorylation site at Thr(89) We found that Mst1 can phosphorylate LPL in vitro and that Mst1 can interact with LPL in cells. Removal of the Mst1 phosphorylation site by mutating Thr(89) to Ala impaired localization of LPL to the actin-rich lamellipodia of T cells. Expression of the T89A LPL mutant failed to restore migration of LPL-deficient T cells in vitro. Furthermore, expression of T89A LPL in LPL-deficient hematopoietic cells, using bone marrow chimeras, failed to rescue the phenotype of decreased thymic egress. These results identify LPL as a key effector of Mst1 and establish a novel mechanism linking a signaling intermediate to an actin-binding protein critical to T cell migration.
Assuntos
Movimento Celular , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Animais , Proteínas do Citoesqueleto , Citometria de Fluxo , Ativação Linfocitária , Linfócitos/imunologia , Camundongos , Proteínas dos Microfilamentos , Fosfoproteínas/deficiência , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Pseudópodes/imunologia , Pseudópodes/fisiologiaRESUMO
This study aimed to co-culture Jurkat T lymphocytes with inactivated Mycobacterium tuberculosis (Mtb H37Ra), explore whether T lymphocytes could phagocytose H37Ra cells, and determine the underlying mechanism. Jurkat T lymphocytes were co-cultured with H37Ra cells, and confocal laser scanning microscopy, electron microscopy, and flow cytometry techniques were used to identify phagocytosis and elucidate its mechanism. After Jurkat T lymphocytes phagocytosed H37Ra cells, the cell body became larger, with abundant cytoplasm, the portion of the nucleus closest to the bacterium deformed, long and short pseudopodia were extended, and the folds of the cell membrane formed depressions that created phagocytic vesicles surrounding the bacterium. The macropinocytosis inhibitor amiloride and the cytoskeletal inhibitor cytochalasin D were found to inhibit phagocytic efficacy; serum complements might enhance phagocytosis through opsonization. Jurkat T lymphocytes could actively phagocytose inactivated Mtb via the macropinocytotic mechanism. Actin remodeling played an important role in the macropinocytotic process. Serum complements may regulate phagocytosis.
Assuntos
Mycobacterium tuberculosis/imunologia , Fagocitose , Fagossomos/imunologia , Pseudópodes/imunologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Amilorida/farmacologia , Técnicas de Cocultura , Proteínas do Sistema Complemento/farmacologia , Citocalasina D/farmacologia , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Temperatura Alta , Humanos , Células Jurkat , Microscopia Eletrônica , Mycobacterium tuberculosis/ultraestrutura , Inibidores da Síntese de Ácido Nucleico/farmacologia , Proteínas Opsonizantes/farmacologia , Fagossomos/efeitos dos fármacos , Fagossomos/microbiologia , Fagossomos/ultraestrutura , Pseudópodes/efeitos dos fármacos , Pseudópodes/microbiologia , Pseudópodes/ultraestruturaRESUMO
CD147 is expressed at low levels in normal tissues but frequently highly expressed in a wide range of tumor types such as lung, breast, and liver and therefore it is a potentially unique therapeutic target for these diverse tumor types. We previously generated a murine antibody HAb18 which suppresses matrix met al.loproteinase-2 and matrix metalloproteinase-9 secretion, attenuates cell invasion by blocking the CD147 molecule in tumor cells. Here, we generated a chimeric antibody containing the variable heavy and variable light chains of murine HAb18 and the constant regions of human IgG1γ1 and human κ chain as a potential therapeutic agent (designated cHAb18). Quantitative measurement of cHAb18 antibody affinity for antigen CD147 with surface plasmon resonance showed the equilibrium dissociation constant KD was 2.66 × 10(-10) mol/L, similar to that of KD 2.73 × 10(-10) mol/L for murine HAb18. cHAb18 induced antibody-dependent cell-mediated cytotoxicity in two hepatocellular carcinoma cell lines, SMMC-7721 and Huh-7 cells. It inhibited cancer invasion and migration in hepatocellular carcinoma cells by specifically blocking CD147. Except for the depression of matrix metalloproteinase-2 and matrix metalloproteinase-9 expressions, cHAb18 antibody suppressed cell motility by rearrangement of actin cytoskeleton, which was probably induced by decreasing the phosphorylation of focal adhesion kinase, phosphatidylinositide-3 kinase (PI3K), Akt, and Girdin in the integrin signaling pathway. In an orthotopic model of hepatocellular carcinoma in BALB/c nude mice, cHAb18 treatment effectively reduced the tumor metastasis in liver and prolonged the survival. These findings reveal new therapeutic potential for cHAb18 antibody targeting CD147 on tumor therapy.
Assuntos
Anticorpos/uso terapêutico , Basigina/imunologia , Carcinoma Hepatocelular/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Anticorpos/genética , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Células CHO , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/secundário , Linhagem Celular Tumoral , Cricetulus , Feminino , Quinase 1 de Adesão Focal/metabolismo , Células Hep G2 , Humanos , Ácidos Hidroxâmicos/farmacologia , Imunoglobulina G/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Neoplasias Hepáticas/patologia , Metaloproteinase 2 da Matriz/imunologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/imunologia , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos/metabolismo , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pseudópodes/imunologia , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais , Anticorpos de Cadeia Única/imunologia , Fibras de Estresse/imunologia , Proteínas de Transporte Vesicular/metabolismoRESUMO
Activated macrophages play an important role in both innate and adaptive immune responses, and aberrant activation of macrophages often leads to inflammatory and immune disorders. However, the molecular mechanisms of how macrophages are activated are not fully understood. In this study, we identify a novel role for histone deacetylse 6 (HDAC6) in lipopolysaccharide (LPS)-induced macrophage activation. Our data show that suppression of HDAC6 activity significantly restrains LPS-induced activation of macrophages and production of pro-inflammatory cytokines. Further study reveals that the regulation of macrophage activation by HDAC6 is independent of F-actin polymerization and filopodium formation; instead, it is mediated by the effects of HDAC6 on cell adhesion and microtubule acetylation. These data thus suggest that HDAC6 is an important regulator of LPS-induced macrophage activation and might be a potential target for the management of inflammatory disorders.
Assuntos
Histona Desacetilases/metabolismo , Imunidade Inata , Inflamação/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Acetilação , Actinas/imunologia , Actinas/metabolismo , Animais , Adesão Celular/genética , Citocinas/imunologia , Citocinas/metabolismo , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases/administração & dosagem , Histona Desacetilases/imunologia , Inflamação/genética , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/genética , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Microtúbulos/imunologia , Microtúbulos/metabolismo , Pseudópodes/genética , Pseudópodes/imunologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Natural Killer (NK) cells are highly mobile, specialized sub-populations of lymphocytic cells that survey their host to identify and eliminate infected or tumor cells. They are one of the key players in innate immunity and do not need prior activation through antigen recognition to deliver cytotoxic packages and release messenger chemicals to recruit immune cells. Cytohesin associated scaffolding protein (CASP) is a highly expressed lymphocyte adaptor protein that forms complexes with vesicles and sorting proteins including SNX27 and Cytohesin-1. In this study we show that by using stably integrated shRNA, CASP has a direct role in the secretion of IFN-γ, and NK cell motility and ability to kill tumor cells. CASP polarizes to the leading edge of migrating NK cells, and to the immunological synapse when engaged with tumor cells. However, CASP is not associated with cytotoxic granule mediated killing. CASP is a multi-faceted protein, which has a very diverse role in NK cell specific immune functions.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Interferon gama/biossíntese , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/fisiologia , Degranulação Celular , Linhagem Celular , Movimento Celular/imunologia , Movimento Celular/fisiologia , Polaridade Celular/imunologia , Polaridade Celular/fisiologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Citotoxicidade Imunológica , Proteínas de Ligação a DNA , Técnicas de Silenciamento de Genes , Humanos , Imunidade Inata , Sinapses Imunológicas/fisiologia , Células K562 , Pseudópodes/imunologia , Pseudópodes/fisiologia , RNA Interferente Pequeno/genética , Receptores CXCR4/fisiologia , Fatores de TranscriçãoRESUMO
Cartilage destruction mediated by invasive fibroblast-like synoviocytes (FLS) plays a central role in pathogenesis of rheumatoid arthritis (RA). Increased cell migration and degradation of extracellular matrix are fundamental to these processes. The class I PI3Ks control cell survival, proliferation, and migration, which might be involved in cartilage damage in RA. PI3Kδ isoform was recently identified as a key regulator of FLS growth and survival, suggesting that it could contribute to synoviocyte aggressive behavior. Therefore, we assessed the role of PI3Kδ in RA synoviocyte migration and invasion. We observed that PI3Kδ inhibition or small interfering RNA knockdown decreased platelet-derived growth factor (PDGF)-mediated migration and invasion of FLS. We then showed that PI3Kδ regulates the organization of actin cytoskeleton and lamellipodium formation during PDGF stimulation. To gain insight into molecular mechanisms, we examined the effect of PI3Kδ inhibition on Rac1/PAK, FAK, and JNK activation. Our studies suggest that Rac1/PAK is key target of PDGF-mediated PI3Kδ signaling, whereas FAK and JNK are not involved. Thus, PI3Kδ contributes to multiple aspects of the pathogenic FLS behavior in RA. These observations, together with previous findings that PI3Kδ regulates FLS growth and survival, suggest that PI3Kδ inhibition could be chondroprotective in RA by modulating synoviocyte growth, migration, and invasion.
Assuntos
Artrite Reumatoide/imunologia , Movimento Celular/imunologia , Fibroblastos/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Membrana Sinovial/imunologia , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/imunologia , Citoesqueleto de Actina/patologia , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Movimento Celular/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Classe I de Fosfatidilinositol 3-Quinases , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Feminino , Fibroblastos/patologia , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/imunologia , Humanos , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/imunologia , Masculino , Fosfatidilinositol 3-Quinases/genética , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/imunologia , Pseudópodes/genética , Pseudópodes/imunologia , Pseudópodes/patologia , Membrana Sinovial/patologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/imunologiaRESUMO
Phagocytes clear the body of undesirable particles such as infectious agents and debris. To extend pseudopods over the surface of targeted particles during engulfment, cells must change shape through extensive membrane and cytoskeleton remodeling. We observed that pseudopod extension occurred in two phases. In the first phase, pseudopods extended rapidly, with actin polymerization pushing the plasma membrane forward. The second phase occurred once the membrane area from preexisting reservoirs was depleted, leading to increased membrane tension. Increased tension directly altered the small Rho GTPase Rac1, 3'-phosphoinositide, and cytoskeletal organization. Furthermore, it activated exocytosis of vesicles containing GPI-anchored proteins, increasing membrane area and phagocytosis efficiency for large particles. We thus propose that, during phagocytosis, membrane remodeling, cytoskeletal organization, and biochemical signaling are orchestrated by the mechanical signal of membrane tension. These results put a simple mechanical signal at the heart of understanding immunological responses.
Assuntos
Actinas/metabolismo , Membrana Celular/imunologia , Fagocitose/imunologia , Pseudópodes/imunologia , Animais , Proteínas de Bactérias , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Citoesqueleto/fisiologia , Transferência Ressonante de Energia de Fluorescência , Histidina/análogos & derivados , Histidina/metabolismo , Proteínas Luminescentes , Camundongos , Microscopia Confocal/métodos , Pinças Ópticas , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
B cells acquire membrane-bound cognate antigens from the surface of the APCs by forming an IS, similar to that seen in T cells. Recognition of membrane-bound antigens on the APCs initiates adhesion of B lymphocytes to the antigen-tethered surface, which is followed by the formation of radial lamellipodia-like structures, a process known as B cell spreading. The spreading response requires the rearrangement of the submembrane actin cytoskeleton and is regulated mainly via signals transmitted by the BCR. Here, we show that cytoplasmic calcium is a regulator of actin cytoskeleton dynamics in B lymphocytes. We find that BCR-induced calcium mobilization is indispensible for adhesion and spreading of B cells and that PLCγ and CRAC-mediated calcium mobilization are critical regulators of these processes. Measuring calcium and actin dynamics in live cells, we found that a generation of actin-based membrane protrusion is strongly linked to the dynamics of a cytoplasmic-free calcium level. Finally, we demonstrate that PLCγ and CRAC channels regulate the activity of actin-severing protein cofilin, linking BCR-induced calcium signaling to the actin dynamics.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/imunologia , Actinas/genética , Actinas/imunologia , Animais , Apresentação de Antígeno , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/imunologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Cofilina 1/genética , Cofilina 1/imunologia , Cofilina 1/metabolismo , Regulação da Expressão Gênica/imunologia , Vetores Genéticos , Lentivirus/genética , Camundongos , Fosfolipase C gama/genética , Fosfolipase C gama/imunologia , Fosfolipase C gama/metabolismo , Pseudópodes/imunologia , Pseudópodes/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais , Transdução GenéticaRESUMO
Cell-mediated transmission and dissemination of sexually-acquired human immunodeficiency virus 1 (HIV-1) in the host involves the migration of immature dendritic cells (iDCs). iDCs migrate in response to the HIV-1 envelope protein, gp120, and inhibiting such migration may limit the mucosal transmission of HIV-1. In this study, we elucidated the mechanism of HIV-1-gp120-induced transendothelial migration of iDCs. We found that gp120 enhanced the binding of Wiskott-Aldrich Syndrome protein (WASp) and the Actin-Related Protein 2/3 (Arp2/3) complex with ß-actin, an interaction essential for the proper formation of podosomes, specialized adhesion structures required for the migration of iDCs through different tissues. We further identified Leukocyte-Specific Protein 1 (LSP1) as a novel component of the WASp-Arp2/3-ß-actin complex. Pretreating iDCs with an active fragment of the secretory glycoprotein Slit2 (Slit2N) inhibited HIV-1-gp120-mediated migration and podosome formation, by inducing the cognate receptor Roundabout 1 (Robo1) to bind to and sequester WASp and LSP1 from ß-actin. Slit2N treatment also inhibited Src signaling and the activation of several downstream molecules, including Rac1, Pyk2, paxillin, and CDC42, a major regulator of podosome formation. Taken together, our results support a novel mechanism by which Slit2/Robo1 may inhibit the HIV-1-gp120-induced migration of iDCs, thereby restricting dissemination of HIV-1 from mucosal surfaces in the host.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Proteínas dos Microfilamentos/imunologia , Proteínas do Tecido Nervoso/imunologia , Receptores Imunológicos/imunologia , Proteína da Síndrome de Wiskott-Aldrich/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/imunologia , Actinas/metabolismo , Western Blotting , Células Cultivadas , Células Dendríticas/metabolismo , Quinase 2 de Adesão Focal/imunologia , Quinase 2 de Adesão Focal/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Proteínas do Tecido Nervoso/metabolismo , Paxilina/imunologia , Paxilina/metabolismo , Ligação Proteica/imunologia , Proteínas Proto-Oncogênicas pp60(c-src)/imunologia , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Pseudópodes/imunologia , Interferência de RNA , Receptores Imunológicos/metabolismo , Transdução de Sinais/imunologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/imunologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas RoundaboutRESUMO
In larvae of the starfish, Asterina pectinifera, mesenchyme cells operate in the defense system through various behaviors. We have investigated mesenchyme cell dynamics during the immune response by identifying ApDOCK, a new member of the DOCK180 superfamily protein. In 4-day-old bipinnaria larvae processed for morpholino oligonucleotide-mediated knockdown of ApDOCK, injection of inorganic foreign substances revealed that (1) mesenchyme cells fail to undergo either directed migration toward a large oil-droplet or persistent spreading on the oil-droplet after contact; (2) neither uptake of micro-beads nor cell-to-cell fusion on the large oil-droplet differed from that of mesenchyme cells from control larvae. Similar behaviors were also recorded in experiments where bacteria were injected. Under culture conditions, the expression level of ApDOCK mRNA was significantly associated with the immunological behavior of mesenchyme cells. Apparently, the mesenchyme cells from ApDOCK loss-of-function larvae exhibited insufficient lamellipodium formation via lack of fibrous form of actin organization at the leading edge. These results suggest that the migratory congregation and persistence of encapsulation of larval mesenchyme cells are intracellularly regulated by ApDOCK protein, and this regulation is associated with organization of cytoskeletal actin.