RESUMO
FOXP3+CD4+ regulatory T cells (Tregs) are critical for immune homeostasis and respond to local tissue cues, which control their stability and function. We explored here whether developmental endothelial locus-1 (DEL-1), which, like Tregs, increases during resolution of inflammation, promotes Treg responses. DEL-1 enhanced Treg numbers and function at barrier sites (oral and lung mucosa). The underlying mechanism was dissected using mice lacking DEL-1 or expressing a point mutant thereof, or mice with T cell-specific deletion of the transcription factor RUNX1, identified by RNA sequencing analysis of the DEL-1-induced Treg transcriptome. Specifically, through interaction with αvß3 integrin, DEL-1 promoted induction of RUNX1-dependent FOXP3 expression and conferred stability of FOXP3 expression upon Treg restimulation in the absence of exogenous TGF-ß1. Consistently, DEL-1 enhanced the demethylation of the Treg-specific demethylated region (TSDR) in the mouse Foxp3 gene and the suppressive function of sorted induced Tregs. Similarly, DEL-1 increased RUNX1 and FOXP3 expression in human conventional T cells, promoting their conversion into induced Tregs with increased TSDR demethylation, enhanced stability, and suppressive activity. We thus uncovered a DEL-1/αvß3/RUNX1 axis that promotes Treg responses at barrier sites and offers therapeutic options for modulating inflammatory/autoimmune disorders.
Assuntos
Proteínas de Ligação ao Cálcio/imunologia , Moléculas de Adesão Celular/imunologia , Integrina beta3/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Proteínas de Ligação ao Cálcio/genética , Moléculas de Adesão Celular/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Integrina beta3/genética , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Linfócitos T Reguladores/patologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/imunologia , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/imunologiaRESUMO
Extracellular matrix (ECM) proteins secreted by blood-brain barrier (BBB) endothelial cells (ECs) are implicated in cell trafficking. We discovered that the expression of ECM epidermal growth factor-like protein 7 (EGFL7) is increased in the CNS vasculature of patients with multiple sclerosis (MS), and in mice with experimental autoimmune encephalomyelitis (EAE). Perivascular CD4 T lymphocytes colocalize with ECM-bound EGFL7 in MS lesions. Human and mouse activated T cells upregulate EGFL7 ligand αvß3 integrin and can adhere to EGFL7 through integrin αvß3. EGFL7-knockout (KO) mice show earlier onset of EAE and increased brain and spinal cord parenchymal infiltration of T lymphocytes. Importantly, EC-restricted EGFL7-KO is associated with a similar EAE worsening. Finally, treatment with recombinant EGFL7 improves EAE, reduces MCAM expression, and tightens the BBB in mouse. Our data demonstrate that EGFL7 can limit CNS immune infiltration and may represent a novel therapeutic avenue in MS.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Fatores de Crescimento Endotelial/genética , Medula Espinal/efeitos dos fármacos , Animais , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Antígeno CD146/genética , Antígeno CD146/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Proteínas de Ligação ao Cálcio , Família de Proteínas EGF , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Fatores de Crescimento Endotelial/deficiência , Fatores de Crescimento Endotelial/imunologia , Fatores de Crescimento Endotelial/farmacologia , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/imunologia , Feminino , Regulação da Expressão Gênica , Humanos , Integrina alfa5/genética , Integrina alfa5/imunologia , Integrina beta3/genética , Integrina beta3/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Knockout , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Medula Espinal/imunologiaRESUMO
The control of complement activation within embryo-endometrium environment is critical for embryo survival. Cell evasion from complement attack requires interaction of complement regulatory proteins (CRPs) with cell adhesion αvß3 integrin. We aim to compare the expression of CRPs in endometria of women with and without endometriosis and to examine the molecular interaction of decay accelerating factor (DAF) with αvß3 integrin. Endometrial expression of Membrane cofactor protein (CD46), Decay accelerating factor (DAF), Membrane attack complex inhibitory factor (CD59) and ß3 integrin subunit were determined through menstrual cycle by immunohistochemistry. DAF protein quantity was determined by Western blot and mRNA levels measured in epithelial cells isolated by laser capture microdissection (LCM). Using in vitro assay, we examined DAF and ß3 integrin expression through paracrine regulation between endometrial compartments. To determine whether ß3 integrin and DAF interacts in vivo, endometrial samples were subjected to immunoprecipitation and colocalization using dual immunofluorescence technique. DAF and ß3 integrin expression were significantly low in samples from women with endometriosis during mid secretory phase. This observation was supported by decreased DAF protein quantity; faint DAF and ß3 integrin interaction and reduced mRNA levels in cells dissected by LCM. Moreover epithelial DAF and ß3 integrin expression through paracrine regulation by progesterone from stromal compartment was disrupted in endometriosis. Endometria from women with endometriosis exhibits aberrant expression of complement proteins. The abnormal DAF expression potentially compromises embryo survival, contributing to understand the implantation failure in women with endometriosis.
Assuntos
Antígenos CD55/metabolismo , Proteínas do Sistema Complemento/imunologia , Endometriose/imunologia , Endométrio/imunologia , Integrina beta3/metabolismo , Adulto , Biópsia , Antígenos CD55/imunologia , Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Implantação do Embrião/imunologia , Endometriose/patologia , Endométrio/patologia , Feminino , Humanos , Integrina beta3/imunologia , Ciclo Menstrual/imunologia , Comunicação Parácrina/imunologia , Progesterona/metabolismoRESUMO
For many years, splenectomy has been used to treat immune thrombocytopenia (ITP), and this procedure benefits approximately two-thirds of the treated patients. Although splenectomy may raise platelet counts, antibody-coated platelets and cytotoxic T lymphocytes appear to persist or can change over time. To better understand how the spleen may affect anti-platelet immune responses, we used a murine model of ITP demonstrating both antibody-mediated and T lymphocyte-mediated thrombocytopenia. Mice with severe combined immunodeficiency (SCID) were either splenectomized or not and transfused with splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61(+) platelets. Platelet counts and anti-platelet antibody levels were performed weekly. After 4 weeks, the mice were sacrificed, and lymphoid organs were harvested and examined by flow cytometry to quantify CD4(+)CD25(+)FoxP3(+) Tregs and conventional cross-presenting XCR1(+) and tolerizing SIRPα+ dendritic cells. The results indicate that compared with control non-splenectomized mice, thrombocytopenia was improved and anti-platelet antibody production was significantly diminished in all splenectomized mice that received immune splenocytes. Splenectomized SCID mice also had a marked reduction in Tregs in the thymus together with an increased proportion of both thymic dendritic cell subsets that correlated with increased platelet counts. Of interest, although splenectomy diminished anti-platelet antibody production and raised platelet counts, marrow megakaryocyte densities were still significantly reduced in mice that received immune splenocytes. These results suggest that the spleen in murine ITP not only is the primary site responsible for platelet destruction, but it also controls, to a significant extent, the antibody response against platelets and the migration patterns of lymphocyte subsets.
Assuntos
Autoanticorpos/imunologia , Plaquetas/imunologia , Linfócitos/imunologia , Púrpura Trombocitopênica Idiopática/imunologia , Baço/imunologia , Animais , Biomarcadores , Medula Óssea/patologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Integrina beta3/genética , Integrina beta3/imunologia , Contagem de Leucócitos , Linfócitos/metabolismo , Linfócitos/patologia , Megacariócitos/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Contagem de Plaquetas , Púrpura Trombocitopênica Idiopática/metabolismo , Púrpura Trombocitopênica Idiopática/patologia , Púrpura Trombocitopênica Idiopática/terapia , Baço/patologia , Esplenectomia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Timócitos/imunologia , Timócitos/metabolismoRESUMO
AIM: OLT is the best alternative for patients with end-stage liver diseases. However, as the need for organs surpasses donor availability, alternatives to OLT are required. LCT could be a useful option versus OLT in several patients even though its low cell-engraftment hampers its efficiency. Endothelial cell barrier is the main obstacle for the implantation of cells into the parenchyma. Our study has focused on the modification of the endothelial barrier with monoclonal antibodies against adhesion molecules in order to increase cell engraftment in a mouse model of liver cell transplantation. METHODS: Anti-mouse CD54 and anti-mouse CD61 antibodies were administered intrasplenically to healthy mice within 60 min prior to stem cell transplantation. Animals were sacrificed either short term at 2h or middle term seven days after transplantation. Immunohistochemical techniques to detect alkaline phosphatase activity were used to identify the transplanted cells within the liver parenchyma. RESULTS: Anti-CD54 and anti-CD61 administration increases vascular patency and cell engraftment. This represents a 32% and 45% increase, respectively, of engrafted cells compared to the control (p<0.05). CONCLUSION: Modification of the vascular wall with monoclonal antibodies against endothelial adhesion molecules before cell transplantation enhances cell engraftment into the mouse liver.
Assuntos
Anticorpos/farmacologia , Doença Hepática Terminal/terapia , Sobrevivência de Enxerto/efeitos dos fármacos , Integrina beta3/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Fígado/imunologia , Transplante de Células-Tronco , Aloenxertos , Animais , Modelos Animais de Doenças , Doença Hepática Terminal/imunologia , Sobrevivência de Enxerto/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Masculino , CamundongosRESUMO
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia, which is caused by maternal antibodies against ß3 integrin and occasionally by maternal antibodies against other platelet antigens, such as glycoprotein GPIbα, has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately explored. Utilizing murine models of FNAIT and a high-frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-ß3 integrin-mediated, but not anti-GPIbα-mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti-ß3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis, all of which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis were further reproduced in neonates by injection of anti-ß3 integrin, but not anti-GPIbα antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and AKT phosphorylation were inhibited only by murine anti-ß3 integrin antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest that fetal hemostasis is distinct and that impairment of angiogenesis rather than thrombocytopenia likely causes FNAIT-associated ICH. Additionally, our results indicate that maternal IVIG therapy can effectively prevent this devastating disorder.
Assuntos
Antígenos de Plaquetas Humanas/imunologia , Autoantígenos/imunologia , Plaquetas/imunologia , Imunidade Materno-Adquirida , Imunoglobulina G/imunologia , Imunoglobulinas Intravenosas/uso terapêutico , Integrina beta3/imunologia , Hemorragias Intracranianas/etiologia , Neovascularização Patológica/etiologia , Trombocitopenia Neonatal Aloimune/imunologia , Animais , Especificidade de Anticorpos , Apoptose , Encéfalo/irrigação sanguínea , Encéfalo/embriologia , Modelos Animais de Doenças , Feminino , Sangue Fetal/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Soros Imunes/toxicidade , Integrina beta3/genética , Hemorragias Intracranianas/embriologia , Hemorragias Intracranianas/imunologia , Hemorragias Intracranianas/fisiopatologia , Masculino , Troca Materno-Fetal , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/imunologia , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologia , Gravidez , Proteínas Proto-Oncogênicas c-akt/fisiologia , Vasos Retinianos/embriologia , Vasos Retinianos/patologia , Trombocitopenia Neonatal Aloimune/embriologia , Trombocitopenia Neonatal Aloimune/prevenção & controleRESUMO
Integrins are a large family of heterodimeric proteins that are involved in cell adhesion, migration, and proliferation. Integrin diversity and function is regulated by alternative splicing. Membrane-bound and truncated ß3-integrins were shown to be key players in cancer metastasis. However, the immunomodulatory functions of the soluble (s) ß3-integrin have not been investigated yet. In this study, we described a novel form of sß3-integrin in acute myeloid leukaemia (AML) patients. Furthermore, we assessed the role of the sß3-integrin in the modulation of natural killer (NK)-cell activity. Levels of sß3-integrin were analysed in plasma samples of 23 AML patients and 26 healthy donors by ELISA. The capacity of sß3-integrin to regulate NK cell activity was investigated using proliferation, cytokine secretion, and cytotoxicity assays. Circulating sß3-integrin was detected in the plasma of 8 AML patients. NK cells showed significantly higher proliferation rates after stimulation with sß3-integrin and IL-2, IL-15 (73%). Significant increases in the NK cells' secreted levels of TNF-α, IFN-γ were measured in presence of sß3-integrin. In addition, sß3-integrin caused the upregulation of Granzyme B transcripts levels as well as FasL expression levels in NK cells. Most importantly, significantly higher K562 or AML blast target cell lysis rates were observed when NK cells were exposed to sß3-integrin. This study reports the identification of a novel sß3-integrin in AML patients and provides novel insights into its role in the immunomodulation of NK cell activity.
Assuntos
Integrina beta3/imunologia , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/imunologia , Proliferação de Células , Citocinas/imunologia , Proteína Ligante Fas/genética , Regulação Leucêmica da Expressão Gênica , Granzimas/genética , Células HEK293 , Humanos , Integrina beta3/sangue , Células Matadoras Naturais/citologia , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/genética , Isoformas de Proteínas/sangue , Isoformas de Proteínas/imunologia , Ativação Transcricional , Regulação para CimaRESUMO
HIV-1-associated neuroinflammation persists even with effective combined antiretroviral therapy, and it is associated with the presence of activated monocytes/macrophages within the CNS. To infiltrate the CNS, monocytes transmigrate across the selectively permeable blood-brain barrier, which is compromised during HIV-1 infection. Interestingly, platelet-derived excess soluble CD40 ligand found in the plasma and cerebrospinal fluid of HIV-1-infected individuals with cognitive impairment has previously been implicated in increased blood-brain barrier permeability. In this study we show that soluble CD40 ligand also promotes the formation of complexes between inflammatory monocytes and activated platelets (PMCs), which are detected by flow cytometry as monocytes that express excess of CD61, a platelet marker, and that these complexes are increased in individuals with HIV-1 infection. PMCs exhibit an enhanced ability to adhere to human brain microvascular endothelial cells as compared with monocytes alone, and they migrate across the transendothelial barrier. These complexes can be found marginalized in the lumen of postcapillary venules in postmortem brain tissue derived from cases of HIV-1-associated encephalitis. The extravasation of monocytes across the brain endothelium may exacerbate neuroinflammation, indicating that enhancing this event via platelet interaction may be a contributing factor in the development of cognitive impairment. Thus, dampening platelet activation, and in turn PMC formation, with antiplatelet agents may prove beneficial in developing adjunctive therapies for use in combination with combined antiretroviral therapy in an effort to reduce HIV-1-associated neurologic deficit.
Assuntos
Plaquetas/imunologia , Barreira Hematoencefálica/imunologia , Encefalite/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Monócitos/imunologia , Adulto , Plaquetas/patologia , Barreira Hematoencefálica/patologia , Ligante de CD40/imunologia , Circulação Cerebrovascular/imunologia , Encefalite/etiologia , Encefalite/patologia , Células Endoteliais/imunologia , Células Endoteliais/patologia , Feminino , Infecções por HIV/complicações , Infecções por HIV/patologia , Humanos , Integrina beta3/imunologia , Masculino , Pessoa de Meia-Idade , Monócitos/patologiaRESUMO
There is increasing evidence that the effect of chemotherapy on tumor growth is not cell autonomous but relies on the immune system. The objective of this study was therefore to decipher the cellular and molecular mechanisms underlying the role of innate and adaptive immunity in chemotherapy-induced tumor rejection. Treatment of DBA/2 mice bearing P815 mastocytoma with cyclophosphamide induced rejection and long-term protection in a CD4- and CD8-dependent manner. A population of inflammatory-type dendritic cells was dramatically expanded in the lymph nodes of mice that rejected the tumor and correlated with CD4-dependent infiltration, in tumor bed, of tumor-specific CD8+ T lymphocytes. Our data point to a major role of CD4+ T cells in inducing chemokine expression in the tumor, provoking migration of tumor-specific CXCR3+ CD8+ T lymphocytes. Importantly, the analysis of CD8+ T cells specific to P1A/H-2L(d) and P1E/H-2K(d) revealed that cyclophosphamide altered the P815-specific CD8 T repertoire by amplifying the response specific to the mutated P1E antigen.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ciclofosfamida/uso terapêutico , Mastocitoma/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Movimento Celular/imunologia , Proliferação de Células , Células Dendríticas/imunologia , Antígenos H-2/imunologia , Integrina beta3/imunologia , Linfonodos/citologia , Ativação Linfocitária/imunologia , Mastocitoma/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Receptores CXCR3/metabolismoRESUMO
αvß3 integrin represents a novel sensing system which detects herpes simplex virus (HSV) and bacterial constituents. In cooperation with Toll-like receptor 2 (TLR2), it elicits an innate response that leads to activation of type I interferon (IFN), NF-κB, and a specific set of cytokines. We report that this defensive branch is functional in cells which represent experimental models of epithelial, including keratinocytic, and neuronal cells. These are the major targets of HSV in vivo. HSV entered the three cell lines via distinct routes. Hence, the defensive response was independent of the route of virus entry. Soluble gH/gL sufficed to elicit type I IFN and NF-κB activation and represents the viral pathogen-associated molecular pattern (PAMP) of this defense system.
Assuntos
Células Epiteliais/imunologia , Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Integrina alfa5/imunologia , Integrina beta3/imunologia , Interferon Tipo I/imunologia , Glicoproteínas de Membrana/imunologia , Chaperonas Moleculares/imunologia , NF-kappa B/imunologia , Neurônios/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas Virais/imunologia , Linhagem Celular , Contraindicações , Células Epiteliais/virologia , Herpes Simples/genética , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Humanos , Integrina beta3/genética , Interferon Tipo I/genética , Glicoproteínas de Membrana/genética , Chaperonas Moleculares/genética , NF-kappa B/genética , Neurônios/virologia , Proteínas do Envelope Viral/genética , Proteínas Virais/genéticaRESUMO
The role of tissue factor (TF)-containing microparticles in clot propagation has been established, but the ability of circulating microparticles to initiate coagulation has been disputed. However, TF-bearing microparticles, particularly endothelial-microparticles generated during disease, may interact with extracellular matrices which in turn can localise circulating TF to sites of injury. In order to examine this hypothesis in vitro , microparticles were isolated from human coronary artery endothelial cells transfected to overexpress TF, tumour-necrosis factor (TNF) α-treated cells or non-transfected cells lacking TF. The ability of microparticles to bind collagen-IV, fibronectin and fibrin was examined under static conditions and arterial shear rates (650 s⻹), and also in the presence of inhibitory antibodies against ß1-, ß3-, α3- and αv-integrins or an anti-TF antibody. TF-microparticles showed increases of up to 43% and 24% in adherence to collagen-IV and fibronectin, respectively, compared to control microparticles under shear flow. Furthermore, TF-containing microparticles, but not the transfected parent cells had increased levels of ß1-integrin compared to TF-deficient microparticles. Pre-incubation of microparticles with a ß1-integrin-blocking antibody counteracted the additional adhesion of TF-microparticles compared to control microparticles. Finally, adherence of TF microparticles to collagen-IV or fibronectin resulted in increased TF activity by concentrating TF onto the surface. In conclusion, the presence of TF within microparticles enhances the interactions of endothelial cell-derived microparticles with extracellular matrices in an integrin-dependent manner. Accumulation and localisation of these microparticles in turn results in the enhancement of TF activity. This may be an innate mechanism by which TF-bearing microparticles induce coagulation upon vascular injury.
Assuntos
Coagulação Sanguínea , Micropartículas Derivadas de Células/metabolismo , Colágeno Tipo IV/metabolismo , Células Endoteliais/metabolismo , Fibronectinas/metabolismo , Tromboplastina/metabolismo , Anticorpos Bloqueadores/metabolismo , Células Cultivadas , Fibrina/metabolismo , Humanos , Integrina alfa3/imunologia , Integrina alfa3/metabolismo , Integrina alfaV/imunologia , Integrina alfaV/metabolismo , Integrina beta1/imunologia , Integrina beta1/metabolismo , Integrina beta3/imunologia , Integrina beta3/metabolismo , Ligação Proteica , Tromboplastina/genética , Tromboplastina/imunologia , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Heterogeneity, shortage of material, and lack of progenitor-specific cell surface markers are major obstacles to elucidating the mechanisms underlying developmental processes. Here we report a proteomics platform that alleviates these difficulties and demonstrate its effectiveness in fractionating heterogeneous cultures of early endoderm derived from human embryonic stem cells. The approach, designated differential cell-capture antibody array, is based on highly parallel, comparative screening of live cell populations using hundreds of antibodies directed against cell-surface antigens. We used this platform to fractionate the hitherto unresolved early endoderm compartment of CXCR4+ cells and identify several endoderm (CD61+ and CD63+) and non-endoderm (CD271+, CD49F+, CD44+ and B2M+) sub-populations. We provide evidence that one of these sub-populations, CD61+, is directly derived from CXCR4+ cells, displays characteristic kinetics of emergence, and exhibits a distinct gene expression profile. The results demonstrate the potential of the cell-capture antibody array as a powerful proteomics tool for detailed dissection of heterogeneous cellular systems.
Assuntos
Antígenos de Superfície/imunologia , Células-Tronco Embrionárias/química , Células-Tronco Embrionárias/citologia , Endoderma/química , Endoderma/citologia , Proteômica , Receptores CXCR4/análise , Anticorpos/imunologia , Biomarcadores/análise , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Separação Celular , Citometria de Fluxo , Humanos , Receptores de Hialuronatos/análise , Receptores de Hialuronatos/imunologia , Integrina alfa6/análise , Integrina alfa6/imunologia , Integrina beta3/análise , Integrina beta3/imunologia , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Receptores CXCR4/imunologia , Receptores de Fator de Crescimento Neural/análise , Receptores de Fator de Crescimento Neural/imunologia , Tetraspanina 30/análiseRESUMO
We previously reported that patients with early-onset HIV-1 ITP developed a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49-66 epitope. Anti-GPIIIa49-66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)-HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49-66. Since the megakaryocyte (MK) is the progenitor cell for platelets, we have investigated the effect of anti-GPIIIa49-66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49-66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49-66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5-fold in in vitro culture of human cord blood CD34+ cells driven by thrombopoietin (TPO). We also observe a three-fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49-66 antibody. However, we could not detect ROS release in DCFH-loaded mouse megakaryoblastic cells L8057 treated with anti-GPIIIa49-66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of L8057 cells induced by TPO. In fact, we found a dose dependent increase in the percentage of CD41 positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 cells treated with various concentrations of H2O2 (from 5 to 20 µM). We therefore conclude that the anti-GPIIIa49-66 antibody inhibits MK differentiation through ß3 integrin signalling independent of ROS release.
Assuntos
Plaquetas/efeitos dos fármacos , Diferenciação Celular , Infecções por HIV/imunologia , HIV-1/imunologia , Megacariócitos/efeitos dos fármacos , Anticorpos Bloqueadores/farmacologia , Autoanticorpos/farmacologia , Plaquetas/metabolismo , Plaquetas/patologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/fisiologia , HIV-1/patogenicidade , Humanos , Integrina beta3/imunologia , Integrina beta3/metabolismo , Megacariócitos/metabolismo , Megacariócitos/patologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
A novel family of RGD-containing molecules (Tablysin-15) has been molecularly characterised from the salivary gland of the haematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity binding for platelet αIIbß3 and endothelial cell αVß3 integrins, but not for α5ß1 or α2ß1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~1 nM) and marginally to fibronectin (IC50 ~1 µM), but not to collagen. It also inhibits fibroblast growth factor (FGF)-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilised fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that αIIbß3 binds to Tablysin-15. Moreover, immobilised Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin αIIbß3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block αIIbß3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.
Assuntos
Plaquetas/efeitos dos fármacos , Desintegrinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Oligopeptídeos/metabolismo , Inibidores da Angiogênese/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Plaquetas/metabolismo , Plaquetas/patologia , Adesão Celular/efeitos dos fármacos , Proliferação de Células , Dípteros , Desintegrinas/química , Desintegrinas/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Integrina alfa2/imunologia , Integrina alfa2/metabolismo , Integrina alfa5/metabolismo , Integrina beta3/imunologia , Integrina beta3/metabolismo , Oligopeptídeos/química , Oligopeptídeos/genética , Inibidores da Agregação Plaquetária/metabolismo , Ligação Proteica/efeitos dos fármacos , Glândulas Salivares/metabolismo , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/metabolismo , Relação Estrutura-Atividade , ViperidaeRESUMO
The occurrence of transfusion-related alloimmunization against αIIbß3 is a major concern in patients with Glanzmann thrombasthenia (GT). However, few data are available about molecular defects of GT patients with anti-αIIbß3 alloantibodies as well as clinical impact of these antibodies on platelet transfusion. Here, we report a case of type I GT with anti-HLA and anti-αIIbß3 alloantibodies, who underwent laparoscopic total gastrectomy due to gastric cancer. We found a novel ß3 nonsense mutation, 892C > T (Arg272X), and the patient was homozygous for the mutation. Laparoscopic gastrectomy was successfully performed with continuous infusion of HLA-matched platelet concentrates and bolus injection of recombinant factor VIIa at 2 h intervals. Total bleeding was 370 mL and no red-cell transfusion was necessary. Flow cytometric analysis employing anti-αIIbß3 monoclonal antibody revealed that the transfused platelet count was maintained around 20-30 × 109/L during the operation and 10 × 109/L on the following day. Flow cytometric analysis also showed that transfused platelets retained normal reactivity to ADP stimulation. These results indicate that flow cytometry is useful to assess survival and function of transfused platelets in GT patients with anti-αIIbß3 antibodies.
Assuntos
Códon sem Sentido , Integrina beta3 , Isoanticorpos/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Trombastenia/genética , Trombastenia/imunologia , Povo Asiático , Humanos , Integrina beta3/genética , Integrina beta3/imunologia , Japão , Masculino , Pessoa de Meia-Idade , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Trombastenia/terapiaRESUMO
Three dogs of different breeds, ages, and genders were presented with pale mucous membranes, depression, anorexia, and splenomegaly. Observed were severe normocytic, nor-mochromic, nonregenerative anemia, thrombocytopenia, and leukopenia. Blood smears contained large, atypical cells with blue vacuolated cytoplasm, cytoplasmic blebs, round to oval central nuclei, and elevated numbers of cytoplasmic fragment resembling macroplatelets. Bi- and multinucleated atypical cells were found mainly in spleen, lymph nodes, and bone marrow. A final diagnosis of acute megakaryoblastic leukemia (AMegL) was made based on morphology and positivity to the megakaryocyte-derived cell-specific markers von Willebrand factor and CD61. In case nos. 1 and 2, no treatment was initiated, and the dogs died on days 4 and 3, respectively. Case no. 3 received supportive therapy with prednisone, and after a brief improvement the dog died spontaneously 35 days after initial presentation. Only 11 cases of AMegL have been reported in dogs, and the specific diagnostic criteria have not been well established. The presence of vacuolization, cytoplasmic blebs, central round nuclei, cytoplasmic fragments, and multinucleated cells in these three cases were considered useful to differentiate AMegL from other hematopoietic neoplasms.
Assuntos
Doenças do Cão/diagnóstico , Leucemia Megacarioblástica Aguda/veterinária , Animais , Doenças do Cão/sangue , Doenças do Cão/patologia , Cães , Evolução Fatal , Feminino , Integrina beta3/sangue , Integrina beta3/imunologia , Leucemia Megacarioblástica Aguda/sangue , Leucemia Megacarioblástica Aguda/diagnóstico , Leucemia Megacarioblástica Aguda/patologia , Masculino , Fator de von Willebrand/imunologia , Fator de von Willebrand/metabolismoRESUMO
The pathogenesis of childhood chronic immune thrombocytopenic purpura (ITP) is mainly mediated by antiplatelet autoantibodies, which have been shown to induce platelet apoptosis in murine models. Decreased CXCR4 expression, which can regulate apoptotic pathway, has been described in platelet disorders. The present study aims to determine whether platelet apoptosis is increased in pediatric patients with chronic ITP and whether there is any involvement of the CXCR4 chemokines axis. Twenty-one patients and 12 controls were studied. Using flow cytometry, we investigated apoptotic markers of platelets including annexin V, caspase 3, and mitochondrial inner transmembrane potential depolarization. The percentage of the platelets with apoptosis-positive markers was not increased in chronic ITP patients. CXCR4 expression was higher in the patients as detected by flow cytometric (P=0.001) and western blotting analysis (P=0.013). The results also revealed that CXCR4 downstream proteins, Akt phosphorylation was more frequent in chronic ITP patients than controls. Plasma stromal cell-derived factor 1 levels analyzed by enzyme-linked immunosorbent assay were decreased in patients (P=0.001) and inversely correlated to CXCR4 expression (r=-0.62, P<0.001). In conclusion, the study shows platelet apoptosis resistance existing in pediatric patients with chronic ITP. It may be associated with enhanced CXCR4 expression and Akt activation.
Assuntos
Apoptose , Plaquetas/patologia , Regulação da Expressão Gênica , Púrpura Trombocitopênica Idiopática/genética , Receptores CXCR4/genética , Adolescente , Anexina A5/imunologia , Plaquetas/citologia , Plaquetas/imunologia , Caspase 3/imunologia , Criança , Pré-Escolar , Doença Crônica , Citocinas/imunologia , Feminino , Humanos , Integrina beta3/imunologia , Masculino , Potencial da Membrana Mitocondrial , Glicoproteína IIb da Membrana de Plaquetas/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Púrpura Trombocitopênica Idiopática/imunologia , Púrpura Trombocitopênica Idiopática/patologia , Receptores CXCR4/imunologiaRESUMO
Platelets play an essential role in the development of cardiovascular diseases and are the target of several agents that can inhibit their function. Despite the existence of a wide array of techniques to study platelet function, an assay to evaluate several platelet signalling pathways in a high-throughput fashion, combined with minimal blood volume and handling is still needed. We have developed a sensitive assay in the form of a sandwich ELISA where monoclonal antibodies against P-selectin or alphaIIbbeta3 and GPIbalpha were used to capture and detect platelets, respectively, in the presence of five different agonists [ADP, TRAP (thrombin receptor agonist), U46619 (thromboxane A2 analogue), collagen-related-peptide, and arachidonic acid]. Binding of platelets to the antibodies increased dose-dependently with the concentration of either agonist, while binding of ADP-activated platelets was abrogated when inhibitors of platelet activation were concomitantly added. The test showed good sample reproducibility in 15 healthy donors with conserved platelet response to agonists throughout the assay. Healthy subjects could be identified as normal-, hypo- or hyper-responders for each agonist, which for most cases (73%) was confirmed upon retesting. Finally, we demonstrated that the platelet ELISA assay can not only be used in platelet-rich plasma but also in whole blood; it now awaits large scale studies to assess its full screening and diagnostic values.
Assuntos
Ensaio de Imunoadsorção Enzimática , Ensaios de Triagem em Larga Escala , Ativação Plaquetária , Testes de Função Plaquetária , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Difosfato de Adenosina , Alprostadil/farmacologia , Anticorpos Monoclonais , Ácido Araquidônico , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Plaquetas/fisiologia , Proteínas de Transporte , Relação Dose-Resposta a Droga , Citometria de Fluxo , Glicoproteínas/imunologia , Humanos , Imunoglobulinas/imunologia , Integrina alfa2/imunologia , Integrina beta3/imunologia , Variações Dependentes do Observador , Selectina-P/imunologia , Peptídeos , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , S-Nitroso-N-Acetilpenicilamina/farmacologiaRESUMO
Adenoviral vectors used in gene therapy induce inflammation, although the underlying mechanisms are currently unknown. In this issue of Immunity, Di Paolo et al. (2009) implicate interleukin-1 alpha (IL- 1 alpha) in virus-induced inflammation and identify the beta 3 integrin as the key receptor regulating IL-1 alpha activity.
Assuntos
Adenoviridae/imunologia , Vetores Genéticos/imunologia , Inflamação/virologia , Integrina beta3/imunologia , Interleucina-1alfa/imunologia , Animais , Terapia Genética , Imunidade Inata , Inflamação/imunologia , CamundongosRESUMO
The recognition of viral components by host pattern-recognition receptors triggers the induction of the antiviral innate immune response. Toll-like receptor 9 (TLR9) and NLRP3 inflammasome were shown to be the principal specific sensors of viral double-stranded DNA. Here we present evidence that macrophages in vivo activated an innate immune response to a double-stranded DNA virus, adenovirus (Ad), independently of TLR9 or NLRP3 inflammasome. In response to Ad, macrophage-derived IL-1 alpha triggered IL-1RI-dependent production of a defined set of proinflammatory cytokines and chemokines. The IL-1 alpha-mediated response required a selective interaction of virus arginine-glycine-aspartic acid (RGD) motifs with macrophage beta(3) integrins. Thus, these data identify IL-1 alpha-IL-1RI as a key pathway allowing for the activation of proinflammatory responses to the virus, independently of its genomic nucleic acid recognition.