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1.
Cell Host Microbe ; 25(5): 730-745.e6, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31003939

RESUMO

Type I interferon (IFN-I) is critical for antiviral defense, and plasmacytoid dendritic cells (pDCs) are a predominant source of IFN-I during virus infection. pDC-mediated antiviral responses are stimulated upon physical contact with infected cells, during which immunostimulatory viral RNA is transferred to pDCs, leading to IFN production via the nucleic acid sensor TLR7. Using dengue, hepatitis C, and Zika viruses, we demonstrate that the contact site of pDCs with infected cells is a specialized platform we term the interferogenic synapse, which enables viral RNA transfer and antiviral responses. This synapse is formed via αLß2 integrin-ICAM-1 adhesion complexes and the recruitment of the actin network and endocytic machinery. TLR7 signaling in pDCs promotes interferogenic synapse establishment and provides feed-forward regulation, sustaining pDC contacts with infected cells. This interferogenic synapse may allow pDCs to scan infected cells and locally secrete IFN-I, thereby confining a potentially deleterious response.


Assuntos
Antivirais/metabolismo , Adesão Celular , Células Dendríticas/imunologia , Imunidade Inata , Fatores Imunológicos/metabolismo , Interferon Tipo I/metabolismo , Viroses/imunologia , Linhagem Celular , Técnicas de Cocultura , Vírus da Dengue/imunologia , Hepacivirus/imunologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Receptor 7 Toll-Like/metabolismo , Zika virus/imunologia
2.
Methods Mol Biol ; 1930: 1-9, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610592

RESUMO

The inherent ability of T-cells to migrate is critical for a fully functional immune system, both in normal immune surveillance and for mounting an adaptive immune response. At the same time, inappropriate trafficking of T-cells can be a pathological factor for immune-mediated or chronic inflammatory diseases. T-cell motility is critically dependent on a series of ligand-receptor interactions, a precisely regulated intracellular signaling, an involvement of adaptor proteins, and dynamic remodeling of the cytoskeletal systems. The leukocyte integrin LFA-1 receptor present on T-cells binds to the ligand intercellular adhesion molecule 1 (ICAM-1) and this LFA-1/ICAM-1 contact acts as a trigger for T-cell motility. In this book, we present a collection of methods and protocols that are frequently used by researchers to better understand T-cell motility in health and diseases.


Assuntos
Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Domínios e Motivos de Interação entre Proteínas , Linfócitos T/citologia , Movimento Celular , Células Cultivadas , Humanos , Transdução de Sinais , Linfócitos T/metabolismo , Linfócitos T/fisiologia
3.
Methods Mol Biol ; 1930: 19-23, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610594

RESUMO

Regulated migration of T-lymphocytes through high endothelial venules and secondary lymphoid organs is necessary for an adaptive immune response. Uncontrolled trafficking of T-cells is implicated in many pathological conditions, including autoimmune disorders, such as psoriasis and inflammatory bowel disease. T-cell migration is regulated mainly by the αLß2 integrin receptor LFA-1, which interacts primarily with its cognate ligand ICAM-1 expressed on the endothelium. This interaction triggers a plethora of downstream signaling pathways, which are not fully understood. Thus, in order to dissect the signal transduction processes at molecular levels and phenotypic changes in migrating T-cells, a laboratory model mimicking T-cell motility is important. Here, we describe a simple and highly reproducible in vitro model to study T-cell migration.


Assuntos
Movimento Celular , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Domínios e Motivos de Interação entre Proteínas , Linfócitos T/fisiologia , Células Cultivadas , Humanos , Microscopia , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/metabolismo
4.
Methods Mol Biol ; 1930: 25-32, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610595

RESUMO

The exploration screening of phenotypic changes in motile T-cells within a signaling environment has always been an arduous task due to the sheer population of these microscopic cells. In recent years, High-Content Analysis (HCA) has gained epochal momentum and has allowed for a wider range of quantitative multiplexed cell-based assays in the field of lymphocyte signaling. In this chapter, we consolidate our understanding and describe the technical approach and methodology to quantify T-cell migratory phenotypes using HCA. Optimizations to be adopted to generate high-quality cytological images of motile T-cells and subsequent analysis using HCA are detailed as well.


Assuntos
Movimento Celular , Ensaios de Triagem em Larga Escala/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Linfócitos T/fisiologia , Células Cultivadas , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Fenótipo , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/metabolismo
5.
Methods Mol Biol ; 1930: 33-40, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610596

RESUMO

T-lymphocytes are the principle coordinators of the immune defense system and play a major role in the protection of our body against infections, intruders of non-self, and malignancies. To mount an immune response, T-cells need to be effectively employed to tissue sites of infection or inflammation and establish contacts with antigen-presenting cells (APCs) or malignant cells. Understanding how T-cells navigate toward their recruitment sites would offer new therapeutic opportunities. Advancement in the hardware and software upgrades of microscopy technology has created several efficient and easy-to-operate live cell imaging platforms. In this protocol, we present a generalized and simple-to-follow protocol for live cell imaging of migrating T-cells, which can also be adopted to visualize real-time tracking of intracellular signaling events.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Movimento Celular , Rastreamento de Células/métodos , Processamento de Imagem Assistida por Computador/métodos , Ativação Linfocitária , Linfócitos T/fisiologia , Imagem com Lapso de Tempo/métodos , Células Cultivadas , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Microscopia , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia
6.
Methods Mol Biol ; 1930: 41-50, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610597

RESUMO

Visualization of signal transduction events in T-cells has always been a challenge due to their miniscule size. Recent advancement in super-resolution microscopy techniques presents many new opportunities to navigate the spatial and temporal signaling cross-talks in motile T-cells. Here, we provide technical details, optimal conditions, and critical practical considerations that need to be taken into account during cell handling, sample preparation, and image acquisition of motile T-cells for performing three-dimensional structured illumination microscopy (3D-SIM).


Assuntos
Movimento Celular , Rastreamento de Células/métodos , Processamento de Imagem Assistida por Computador/métodos , Imagem Tridimensional/métodos , Iluminação/métodos , Microscopia de Fluorescência/métodos , Linfócitos T/fisiologia , Células Cultivadas , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia
7.
Methods Mol Biol ; 1930: 51-57, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610598

RESUMO

The ability of activated T-lymphocytes to transmigrate toward certain chemokine is one of their characteristic functional properties. Here, we provide step-wise details about an in vitro technique to quantify the kinetics of chemotactic behavior of LFA-1-stimulated T-lymphocytes. The method described herein utilizes a noninvasive electrical impedance-based detection system to monitor T-cell chemotaxis in real-time.


Assuntos
Rastreamento de Células/métodos , Quimiotaxia , Citocinas/metabolismo , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos T/fisiologia , Células Cultivadas , Impedância Elétrica , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia
8.
Methods Mol Biol ; 1930: 91-98, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610603

RESUMO

The immune system and its components defend our body against diverse pathogens and help in maintaining tissue homeostasis. Immune cells are highly dynamic in terms of their growth, migration, differentiation, and effector functions, and adopt diverse metabolic configurations to respond to varying immunological challenges. Growing body of evidence suggests that metabolic pathways fuel immune cells for their functioning, including T-cell migration to the site of infection. This chapter provides detailed methodology for the efficient extraction of T-cell metabolites for successful downstream immunometabolomic profiling of motile T-lymphocytes.


Assuntos
Movimento Celular , Metaboloma , Espectrometria de Massas por Ionização por Electrospray/métodos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células Cultivadas , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Transdução de Sinais , Linfócitos T/citologia
9.
Methods Mol Biol ; 1930: 115-122, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610605

RESUMO

The cycles of internalization of the cell surface ß2 integrin receptor lymphocyte function-associated antigen 1 (LFA-1) and its re-exposure on the plasma membrane are important for T-cell trafficking. Biotinylation of cells enables to measure surface expression of receptors, and after reducing surface biotin with reducing buffer, enables to measure the internalization of receptors. Here, by using biotin in combination with reducing buffer and recombinant intercellular adhesion molecule-1 (rICAM-1)-coated dishes and subsequent Western immunoblot analysis, we describe how to measure internalization of the LFA-1 receptor and its re-expression back to the cell surface in motile T-lymphocytes.


Assuntos
Biotina/metabolismo , Antígenos CD18/metabolismo , Membrana Celular/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Proteínas Recombinantes/metabolismo , Linfócitos T/metabolismo , Biotinilação , Western Blotting , Adesão Celular , Movimento Celular , Humanos
10.
Inflammation ; 42(1): 365-374, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30255285

RESUMO

Luteolin inhibits the adhesion of neutrophils to microvascular endothelial cells and plays an important anti-inflammatory role, owing to its mechanism of suppressing the expression of lymphocyte function-associated antigen-1 (LFA-1) in the neutrophils. Our study deals with the different signaling pathways participating in LFA-1 expression in neutrophils along with the regulation of luteolin in order to elucidate new anti-inflammatory targets of luteolin, thus providing a basis for clinical applications. In our study, neutrophils were separated using density gradient centrifugation and the cAMP levels were determined using ELISA. Additionally, phosphorylation levels of p38 mitogen-activated protein kinase (MAPK), extracellular regulated protein kinase (ERK), phosphatidylinositol-3-kinase (PI3K), and Janus kinase (JAK) were also detected by Western blotting. LFA-1 expression was estimated using flow cytometry. The results showed that inhibiting agents used against p38 MAPK, ERK, PI3K, and JAK could significantly inhibit LFA-1 expression on neutrophils (p < 0.05, p < 0.01). Luteolin also induced a noteworthy elevation of cAMP in neutrophil supernatants (p < 0.01). It could also significantly inhibit ERK phosphorylation (p < 0.05, p < 0.01), and had no obvious effect on p38 MAPK phosphorylation in neutrophils (p > 0.05). However, phosphorylation of PI3K and JAK was not detected in neutrophils. To conclude, the p38 MAPK, ERK, PI3K, and JAK pathways are involved in the regulation of LFA-1 expression in neutrophils, and luteolin partially inhibits LFA-1 expression by increasing cAMP levels and suppressing ERK phosphorylation.


Assuntos
Luteolina/farmacologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Sistema de Sinalização das MAP Quinases , Neutrófilos/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Humanos , Antígeno-1 Associado à Função Linfocitária/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
11.
Immunol Invest ; 48(1): 27-38, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29985717

RESUMO

BACKGROUND: Despite years of research, the treatment of acute kidney injury (AKI) remains a significant challenge. Animal studies presented causal links between elevated regulatory T cell (Treg) response and better prognosis in AKI. Previous studies in mice and humans showed that TIM-3+ Treg cells were more potent than TIM-3- Treg cells. In this study, we investigated the role of TIM-3 in Treg in AKI patients. METHODS: Peripheral blood from AKI patients and healthy controls were gathered, and TIM-3+ Treg subset was examined. RESULTS: Compared to healthy controls, the AKI patients presented a significant upregulation in the frequency of circulating CD4+CD25+ T cells; however, the majority of this increase was from the CD4+CD25+TIM-3- subset, and the frequency of CD4+CD25+TIM-3+ T cells was downregulated in AKI patients. In both healthy controls and AKI patients, the CD4+CD25+TIM-3+ T cells expressed higher levels of Foxp3, and were more potent at expressing LFA-1, LAG-3, CTLA-4, IL-10 and TGF-ß. In addition, the CD4+CD25+TIM-3+ T cells from both healthy controls and AKI patients presented higher capacity to suppress CD4+CD25- T cell proliferation than the CD4+CD25+TIM-3- T cells. Interestingly, the total CD4+CD25+ T cells from AKI patients presented significantly lower inhibitory capacity than those from healthy controls, indicating that the low frequency of CD4+CD25+TIM-3+ T cells was restricting the efficacy of the Treg responses in AKI patients. CONCLUSIONS: We demonstrated that TIM-3 downregulation impaired the function of Treg cells in AKI. The therapeutic potential of CD4+CD25+TIM-3+ T cells in AKI should be investigated in future studies.


Assuntos
Lesão Renal Aguda/imunologia , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Linfócitos T Reguladores/imunologia , Idoso , Animais , Proliferação de Células , Células Cultivadas , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Tolerância Imunológica , Interleucina-10/metabolismo , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Fator de Crescimento Transformador beta/metabolismo
12.
J Labelled Comp Radiopharm ; 62(2): 77-85, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30466143

RESUMO

The drug candidates (2) and (3) are highly potent LFA-1 inhibitors. They were efficiently prepared labeled with carbon-14 using a palladium-catalyzed carboxylation of an iodo-precursor (5) and sodium formate-14 C to afford acid [14 C]-(6), which was coupled via an amide bond to chiral amines (7) and (8) in 52% and 48% overall yield, respectively, and with specific activities higher than 56 mCi/mmol and radiochemical purities of 99%. For stable isotopes synthesis, the amine [2 H8 ]-(7) was synthesized in three steps from 2-cyanopyridine-2 H4 using Kulinkovich-Szymonik aminocyclopropanation, followed by coupling to L-alanine-2,3,3,3-2 H4 -N-t-BOC, and then removal of the BOC-protecting group. Amide bond formation with acid (6) gave [2 H8 ]-(2) in 36% overall yield. The amine [13 C4 ,15 N]-(8) was obtained in two steps using L-threonine-14 C4 ,15 N and then coupled to acid [13 C]-(6) to give [13 C5 ,15 N]-(3) in 56% overall yield.


Assuntos
Radioisótopos de Carbono/química , Antígeno-1 Associado à Função Linfocitária/metabolismo , Compostos Radiofarmacêuticos/síntese química , Ligação Proteica , Compostos Radiofarmacêuticos/farmacologia
13.
Front Immunol ; 9: 2864, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30564247

RESUMO

T cell activation is initiated upon ligand engagement of the T cell receptor (TCR) and costimulatory receptors. The CD28 molecule acts as a major costimulatory receptor in promoting full activation of naive T cells. However, despite extensive studies, why naive T cell activation requires concurrent stimulation of both the TCR and costimulatory receptors remains poorly understood. Here, we explore this issue by analyzing calcium response as a key early signaling event to elicit T cell activation. Experiments using mouse naive CD4+ T cells showed that engagement of the TCR or CD28 with the respective cognate ligand was able to trigger a rise in fluctuating calcium mobilization levels, as shown by the frequency and average response magnitude of the reacting cells compared with basal levels occurred in unstimulated cells. The engagement of both TCR and CD28 enabled a further increase of these two metrics. However, such increases did not sufficiently explain the importance of the CD28 pathways to the functionally relevant calcium responses in T cell activation. Through the autocorrelation analysis of calcium time series data, we found that combined but not separate TCR and CD28 stimulation significantly prolonged the average decay time (τ) of the calcium signal amplitudes determined with the autocorrelation function, compared with its value in unstimulated cells. This increasement of decay time (τ) uniquely characterizes the fluctuating calcium response triggered by concurrent stimulation of TCR and CD28, as it could not be achieved with either stronger TCR stimuli or by co-engaging both TCR and LFA-1, and likely represents an important feature of competent early signaling to provoke efficient T cell activation. Our work has thus provided new insights into the interplay between the TCR and CD28 early signaling pathways critical to trigger naive T cell activation.


Assuntos
Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Sinalização do Cálcio/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Células Apresentadoras de Antígenos , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células COS , Células Cultivadas , Técnicas de Cocultura , Antígeno-1 Associado à Função Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T/imunologia
14.
Sci Signal ; 11(560)2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30538176

RESUMO

T cell entry into inflamed tissue involves firm adhesion, spreading, and migration of the T cells across endothelial barriers. These events depend on "outside-in" signals through which engaged integrins direct cytoskeletal reorganization. We investigated the molecular events that mediate this process and found that T cells from mice lacking expression of the adaptor protein Crk exhibited defects in phenotypes induced by the integrin lymphocyte function-associated antigen 1 (LFA-1), namely, actin polymerization, leading edge formation, and two-dimensional cell migration. Crk protein was an essential mediator of LFA-1 signaling-induced phosphorylation of the E3 ubiquitin ligase c-Cbl and its subsequent interaction with the phosphatidylinositol 3-kinase (PI3K) subunit p85, thus promoting PI3K activity and cytoskeletal remodeling. In addition, we found that Crk proteins were required for T cells to respond to changes in substrate stiffness, as measured by alterations in cell spreading and differential phosphorylation of the force-sensitive protein CasL. These findings identify Crk proteins as key intermediates coupling LFA-1 signals to actin remodeling and provide mechanistic insights into how T cells sense and respond to substrate stiffness.


Assuntos
Actinas/metabolismo , Movimento Celular , Antígeno-1 Associado à Função Linfocitária/metabolismo , Mecanotransdução Celular , Proteínas Proto-Oncogênicas c-crk/metabolismo , Linfócitos T/citologia , Animais , Adesão Celular , Células Cultivadas , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Camundongos Knockout , Linfócitos T/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
15.
Contrast Media Mol Imaging ; 2018: 6508724, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30538613

RESUMO

Atherosclerosis-related morbidity and mortality remain a global concern. Atherosclerotic disease follows a slow and silent progression, and the transition from early-stage lesions to vulnerable plaques remains difficult to diagnose. Inflammation is a key component of the development of atherosclerotic plaque and consequent life-threatening complications. This study assessed 111In-DANBIRT as an in vivo, noninvasive SPECT/CT imaging probe targeting an inflammatory marker, Lymphocyte Function Associated Antigen-1 (LFA-1), in atherosclerotic plaques. Methods. Selective binding of 111In-DANBIRT was assessed using Sprague-Dawley rats exposed to filtered air and ozone (1 ppm) by inhalation for 4 hours to induce a circulating leukocytosis and neutrophilia in peripheral blood. After 24 hours, whole blood was collected and incubated with radiolabeled DANBIRT (68Ga-DANBIRT and 111In-DANBIRT). Isolated cell component smeared slides using cytospin technique were stained with Wright-Giemsa stain. Apolipoprotein E-deficient (apoE-/-) mice were fed either a normal diet or a high-fat diet (HFD) for 8 weeks. Longitudinal SPECT/CT imaging was performed 3 hours after administration at baseline, 4, and 8 weeks of HFD diet, followed by tissue harvesting for biodistribution, serum lipid analysis, and histology. 3D autoradiography was performed in both groups 24 hours after administration of 111In-DANBIRT. Results. Increased specific uptake of radiolabeled DANBIRT by neutrophils in the ozone-exposed group was evidenced by the acute immune response due to 4-hour ozone exposure. Molecular imaging performed at 3 hours using SPECT/CT imaging evidenced an exponential longitudinal increase in 111In-DANBIRT uptake in atherosclerosis lesions in HFD-fed mice compared to normal-diet-fed mice. Such results were consistent with increased immune response to vascular injury in cardiovascular and also immune tissues, correlated by 24 hours after administration of 3D autoradiography. Histologic analysis confirmed atherosclerotic disease progression with an increased vascular lesion area in HFD-fed mice compared to normal-diet-fed mice. Conclusion. 111In-DANBIRT is a promising molecular imaging probe to assess inflammation in evolving atheroma and atherosclerotic plaque.


Assuntos
Aterosclerose/patologia , Radioisótopos de Índio , Inflamação/diagnóstico por imagem , Placa Aterosclerótica/diagnóstico por imagem , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Antígeno-1 Associado à Função Linfocitária/metabolismo , Imagem Molecular/métodos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Ozônio/farmacologia , Ligação Proteica , Compostos Radiofarmacêuticos , Ratos , Ratos Sprague-Dawley
16.
Front Immunol ; 9: 2852, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30568657

RESUMO

The integrin LFA-1 (CD11a/CD18) plays a critical role in the interaction of T cells with antigen presenting cells (APCs) to promote lymphocyte differentiation and proliferation. This integrin can be present either in a closed or in an open active conformation and its activation upon T-cell receptor (TCR) stimulation is a critical step to allow interaction with APCs. In this study we demonstrate that the serine/threonine kinase Ndr2 is critically involved in the initiation of TCR-mediated LFA-1 activation (open conformation) in T cells. Ndr2 itself becomes activated upon TCR stimulation and phosphorylates the intracellular integrin binding partner Filamin A (FLNa) at serine 2152. This phosphorylation promotes the dissociation of FLNa from LFA-1, allowing for a subsequent association of Talin and Kindlin-3 which both stabilize the open conformation of LFA-1. Our data suggest that Ndr2 activation is a crucial step to initiate TCR-mediated LFA-1 activation in T cells.


Assuntos
Filaminas/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos CD18/imunologia , Antígenos CD18/metabolismo , Células Cultivadas , Proteínas do Citoesqueleto/imunologia , Proteínas do Citoesqueleto/metabolismo , Filaminas/genética , Filaminas/imunologia , Células HEK293 , Voluntários Saudáveis , Humanos , Células Jurkat , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mutação , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Fosforilação/imunologia , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas/genética , Receptores de Antígenos de Linfócitos T/imunologia , Serina/metabolismo , Linfócitos T/metabolismo , Talina/imunologia , Talina/metabolismo
17.
PLoS Biol ; 16(11): e2006525, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30427828

RESUMO

Protein transmembrane domains (TMDs) are generally hydrophobic, but our bioinformatics analysis shows that many TMDs contain basic residues at terminal regions. Physiological functions of these membrane-snorkeling basic residues are largely unclear. Here, we show that a membrane-snorkeling Lys residue in integrin αLß2 (also known as lymphocyte function-associated antigen 1 [LFA-1]) regulates transmembrane heterodimer formation and integrin adhesion through ionic interplay with acidic phospholipids and calcium ions (Ca2+) in T cells. The amino group of the conserved Lys ionically interacts with the phosphate group of acidic phospholipids to stabilize αLß2 transmembrane association, thus keeping the integrin at low-affinity conformation. Intracellular Ca2+ uses its charge to directly disrupt this ionic interaction, leading to the transmembrane separation and the subsequent extracellular domain extension to increase adhesion activity. This Ca2+-mediated regulation is independent on the canonical Ca2+ signaling or integrin inside-out signaling. Our work therefore showcases the importance of intramembrane ionic protein-lipid interaction and provides a new mechanism of integrin activation.


Assuntos
Integrinas/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Proteínas de Membrana/fisiologia , Sequência de Aminoácidos , Cálcio/metabolismo , Adesão Celular , Citoplasma/metabolismo , Humanos , Integrinas/metabolismo , Íons , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Proteínas de Membrana/metabolismo , Concentração Osmolar , Ligação Proteica , Conformação Proteica , Domínios Proteicos/fisiologia , Transdução de Sinais , Linfócitos T/metabolismo
18.
Toxins (Basel) ; 10(10)2018 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-30322160

RESUMO

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, has been associated with localized aggressive periodontitis (LAP). In particular, highly leukotoxic strains of A. actinomycetemcomitans have been more closely associated with this disease, suggesting that LtxA is a key virulence factor for A. actinomycetemcomitans. LtxA is secreted across both the inner and outer membranes via the Type I secretion system, but has also been found to be enriched within outer membrane vesicles (OMVs), derived from the bacterial outer membrane. We have characterized the association of LtxA with OMVs produced by the highly leukotoxic strain, JP2, and investigated the interaction of these OMVs with host cells to understand how LtxA is delivered to host cells in this OMV-associated form. Our results demonstrated that a significant fraction of the secreted LtxA exists in an OMV-associated form. Furthermore, we have discovered that in this OMV-associated form, the toxin is trafficked to host cells by a cholesterol- and receptor-independent mechanism in contrast to the mechanism by which free LtxA is delivered. Because OMV-associated toxin is trafficked to host cells in an entirely different manner than free toxin, this study highlights the importance of studying both free and OMV-associated forms of LtxA to understand A. actinomycetemcomitans virulence.


Assuntos
Exotoxinas/metabolismo , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Exotoxinas/toxicidade , Vesículas Extracelulares/metabolismo , Humanos , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/metabolismo , Células THP-1
19.
PLoS One ; 13(10): e0205871, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30335797

RESUMO

The oral bacterium, Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis, as well as systemic infections including endocarditis, produces numerous virulence factors, including a repeats-in-toxin (RTX) protein called leukotoxin (LtxA), which kills human immune cells. The strains of A. actinomycetemcomitans most closely associated with disease have been shown to produce the most LtxA, suggesting that LtxA plays a significant role in the virulence of this organism. LtxA, like many of the RTX toxins, can be divided into four functional domains: an N-terminal hydrophobic domain, which contains a significant fraction of hydrophobic residues and has been proposed to play a role in the membrane interaction of the toxin; the central domain, which contains two lysine residues that are the sites of post-translational acylation; the repeat domain that is characteristic of the RTX toxins, and a C-terminal domain thought to be involved in secretion. In its initial interaction with the host cell, LtxA must bind to both cholesterol and an integrin receptor, lymphocyte function-associated antigen-1 (LFA-1). While both interactions are essential for toxicity, the domains of LtxA involved remain unknown. We therefore undertook a series of experiments, including tryptophan quenching and trypsin digestion, to characterize the structure of LtxA upon interaction with membranes of various lipid compositions. Our results demonstrate that LtxA adopts a U-shaped conformation in the membrane, with the N- and C-terminal domains residing outside of the membrane.


Assuntos
Aggregatibacter actinomycetemcomitans/química , Proteínas de Bactérias/química , Colesterol/química , Proteínas Hemolisinas/química , Antígeno-1 Associado à Função Linfocitária/química , Fatores de Virulência/química , Aggregatibacter actinomycetemcomitans/crescimento & desenvolvimento , Aggregatibacter actinomycetemcomitans/patogenicidade , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Colesterol/metabolismo , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Proteínas Hemolisinas/isolamento & purificação , Proteínas Hemolisinas/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Células Jurkat , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteólise , Tripsina/química , Fatores de Virulência/isolamento & purificação , Fatores de Virulência/metabolismo
20.
Front Immunol ; 9: 2323, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30374350

RESUMO

Despite its excellent efficacy in controlling T cell mediated acute rejection, lymphocyte depletion may promote a humoral response. While T cell repopulation after depletion has been evaluated in many aspects, the B cell response has not been fully elucidated. We tested the hypothesis that the mechanisms also involve skewed T helper phenotype after lymphocytic depletion. Post-transplant immune response was measured from alemtuzumab treated hCD52Tg cardiac allograft recipients with or without anti-LFA-1 mAb. Alemtuzumab induction promoted serum DSA, allo-B cells, and CAV in humanized CD52 transgenic (hCD52Tg) mice after heterotopic heart transplantation. Additional anti-LFA-1 mAb treatment resulted in reduced DSA (Fold increase 4.75 ± 6.9 vs. 0.7 ± 0.5; p < 0.01), allo-specific B cells (0.07 ± 0.06 vs. 0.006 ± 0.002 %; p < 0.01), neo-intimal hyperplasia (56 ± 14% vs. 23 ± 13%; p < 0.05), arterial disease (77.8 ± 14.2 vs. 25.8 ± 20.1%; p < 0.05), and fibrosis (15 ± 23.3 vs. 4.3 ± 1.65%; p < 0.05) in this alemtuzumab-induced chronic antibody-mediated rejection (CAMR) model. Surprisingly, elevated serum IL-21 levels in alemtuzumab-treated mice was reduced with LFA-1 blockade. In accordance with the increased serum IL-21 level, alemtuzumab treated mice showed hyperplastic germinal center (GC) development, while the supplemental anti-LFA-1 mAb significantly reduced the GC frequency and size. We report that the incomplete T cell depletion inside of the GC leads to a systemic IL-21 dominant milieu with hyperplastic GC formation and CAMR. Conventional immunosuppression, such as tacrolimus and rapamycin, failed to reverse AMR, while co-stimulation blockade with LFA-1 corrected the GC hyperplastic response. The identification of IL-21 driven chronic AMR elucidates a novel mechanism that suggests a therapeutic approach with cytolytic induction.


Assuntos
Alemtuzumab/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Interleucinas/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Alemtuzumab/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Doença Crônica , Citocinas/metabolismo , Centro Germinativo/imunologia , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/patologia , Masculino , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo
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