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1.
Cell Death Dis ; 10(11): 860, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31719524

RESUMO

Cell death has a fundamental impact on the evolution of degenerative disorders, autoimmune processes, inflammatory diseases, tumor formation and immune surveillance. Over the past couple of decades extensive studies have uncovered novel cell death pathways, which are independent of apoptosis. Among these is necroptosis, a tightly regulated, inflammatory form of cell death. Necroptosis contribute to the pathogenesis of many diseases and in this review, we will focus exclusively on necroptosis in humans. Necroptosis is considered a backup mechanism of apoptosis, but the in vivo appearance of necroptosis indicates that both caspase-mediated and caspase-independent mechanisms control necroptosis. Necroptosis is regulated on multiple levels, from the transcription, to the stability and posttranslational modifications of the necrosome components, to the availability of molecular interaction partners and the localization of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Accordingly, we classified the role of more than seventy molecules in necroptotic signaling based on consistent in vitro or in vivo evidence to understand the molecular background of necroptosis and to find opportunities where regulating the intensity and the modality of cell death could be exploited in clinical interventions. Necroptosis specific inhibitors are under development, but >20 drugs, already used in the treatment of various diseases, have the potential to regulate necroptosis. By listing necroptosis-modulated human diseases and cataloging the currently available drug-repertoire to modify necroptosis intensity, we hope to kick-start approaches with immediate translational potential. We also indicate where necroptosis regulating capacity should be considered in the current applications of these drugs.

2.
J Photochem Photobiol B ; 185: 169-175, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29936410

RESUMO

Ultraviolet (UV) light is absorbed by nucleic acids, proteins or other endogenous chromophores, such as porphyrins, flavins and melanin, triggering biological processes in skin cells. Both UV-induced mutations in melanocytes and changes in the immune microenvironment are understood to play a role in the development of cutaneous melanoma. The degree of UV-induced stress and the protection against this stress are influenced by both intracellular and intercellular molecular interactions. The present review summarizes the known major molecular biological changes induced by UV light in the skin that play a role in melanoma initiation and promotion. Nevertheless, cutaneous melanoma is not a homogenous disease, and the interaction of variable environmental exposure and different genetic susceptibility and other host factors lead to the formation of melanomas with different biological behavior and clinical characteristics. This review highlights the challenges in the understanding of how UV radiation contributes to the formation of cutaneous melanoma, and reviews the new results of photobiology and their link to tumor genetics and tumor immunology with potential implications on melanoma prevention and therapeutic strategies. The information presented here is expected to add clarity to ongoing research efforts in this field to aid the development of novel strategies to prevent and treat melanoma.


Assuntos
Melanoma/etiologia , Neoplasias Cutâneas/etiologia , Pele/efeitos da radiação , Raios Ultravioleta , Animais , Colecalciferol/química , Colecalciferol/metabolismo , Dano ao DNA/efeitos da radiação , Humanos , Melaninas/química , Melaninas/metabolismo , Melanoma/imunologia , Melanoma/metabolismo , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Pele/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo
3.
Front Immunol ; 9: 151, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29445380

RESUMO

Tumors are composed of abnormally transformed cell types and tissues that differ from normal tissues in their genetic and epigenetic makeup, metabolism, and immunology. Molecular compounds that modulate the immune response against neoplasms offer promising new strategies to combat cancer. Inhibitors targeting the indoleamine-2,3-dioxygenase 1 enzyme (IDO1) represent one of the most potent therapeutic opportunities to inhibit tumor growth. Herein, we assess the biochemical role of IDO1 in tumor metabolism and immune surveillance, and review current diagnostic and therapeutic approaches that are intended to increase the effectiveness of immunotherapies against highly aggressive and difficult-to-treat IDO-expressing cancers.


Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/fisiologia , Neoplasias/enzimologia , Animais , Humanos , Neoplasias/diagnóstico , Neoplasias/terapia
4.
Sci Rep ; 8(1): 1765, 2018 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-29379077

RESUMO

Serotonin is a monoamine neurotransmitter that signals through a wide array of receptors (5-HT1-7) many of which are also involved in immune processes. Dendritic cells (DCs) are crucial players in immune defense by bridging innate and adaptive immune responses via their vast repertoire of pattern recognition receptors and antigen-presenting capability. Although serotonin is known to influence immunity at many levels, cell type-specific expression and function of its receptors remains poorly understood. Here we aimed to study 5-HT1-7 expression and function in CD1a- and CD1a+ human monocyte-derived DCs (moDCs). We found that the 5-HT2B receptor-subtype is solely expressed by the inflammatory CD1a+ moDC subset. Specific 5-HT2B activation potently inhibited TLR2, TLR3, and TLR7/8-induced proinflammatory cytokine and chemokine (TNF-α, IL-6, IL-8, IP-10, IL-12) but not type I interferon-ß responses. 5-HT2B agonism also interfered with the polarization of CD1a+ moDC-primed CD4+ T cells towards inflammatory Th1 and Th17 effector lymphocytes. Here we report the subset-specific expression and immunomodulatory function of 5-HT2B in human moDCs. Our results expand the biological role of 5-HT2B which may act not only as a neurotransmitter receptor, but also as an important modulator of both innate and adaptive immune responses.


Assuntos
Células Dendríticas/imunologia , Fatores Imunológicos/imunologia , Receptor 5-HT2B de Serotonina/imunologia , Antígenos CD1/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/imunologia , Humanos , Monócitos/imunologia , Transdução de Sinais/imunologia , Células Th17/imunologia
5.
Immunol Lett ; 193: 42-50, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29175315

RESUMO

Efficient adjuvants have the potential to trigger both innate and adaptive immune responses simultaneously. Flagellin is a unique pathogen-derived protein, which is recognized by pattern recognition receptors (PRRs) as well as by B-cell and T cell receptors thus providing an important link between innate and adaptive immunity. The aforementioned properties define flagellin as an optimal adjuvant. The induction of immunogenic cell death could be an additional expectation for adjuvants in the context of cancer immunotherapy due to their ability to activate dendritic cells (DC) to present tumor antigens through the engulfment of dying cells. The immunostimulatory potential of flagellin in the course of DC and lymphocyte activation is well documented, however the exact mechanism is not fully explored. Based on this limitation we sought to investigate the potential modulatory effects of flagellin on various cell death processes knowing that it plays detrimental roles in regulating the final outcome of various types of immune responses. Here we provide evidence that the pre-treatment of Jurkat T-cells with recombinant flagellin is able to increase the degree of cell death provoked by FasL or TNF-α, and concomitantly increases the cytotoxic potential of phytohemagglutinin activated T-lymphocytes in a TLR5 dependent way. In contrast to these flagellin-mediated effects on the death receptor-induced signaling events, the mitochondrial apoptotic pathway remained unaffected. Furthermore, the cell culture supernatant of wild type Salmonella enteritidis bacteria, but not their flagellin deficient variant, was able to enhance the Fas-induced cell death process. To define the molecular mechanisms of flagellin-mediated elevated levels of cell death we were able to detect the upregulation of RIP1-dependent signaling events. These findings demonstrate that the cooperative actions of pattern recognition and different death receptors are able to initiate the cell death process with the mobilization of RIP-dependent cell death modalities. This finding highlights the capability of flagellin to act as a potential adjuvant which is relevant for tumor immunotherapy.


Assuntos
Adjuvantes Imunológicos , Flagelina/metabolismo , Receptores de Morte Celular/metabolismo , Salmonella enteritidis/genética , Linfócitos T/imunologia , Imunidade Adaptativa , Apoptose , Células Dendríticas/fisiologia , Proteína Ligante Fas/metabolismo , Flagelina/genética , Humanos , Imunidade Inata , Células Jurkat , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais
6.
Cell Mol Immunol ; 14(4): 380-391, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26521691

RESUMO

The cytoplasmic nucleotide oligomerization domain 2 (NOD2) receptor recognizes the bacterial cell wall component muramyl dipeptide (MDP). NOD2 ligation initiates the nuclear factor kappa B and the mitogen-activated protein kinase cascades. However, administering MDP alone is insufficient to elicit strong cytokine responses in various immune cells, including dendritic cells (DCs). Because the simultaneous presence of various microbial products and cytokines in inflamed tissues modulates DC function, we initiated this study to examine how interferon gamma (IFNγ), a central modulator of inflammation, affects the NOD2-mediated signaling pathway in human conventional DCs (cDCs). Synergistic stimulation of DCs with MDP and IFNγ increased the expression of CD40, CD80, CD83, CD86, and human leukocyte antigen DQ proteins and significantly elevated the production of pro-inflammatory cytokines IL-1ß, IL-6, IL-12, and tumour necrosis factor (TNF), as well as anti-inflammatory cytokine IL-10. Furthermore, the simultaneous presence of MDP and IFNγ was necessary to decrease IkBα protein levels. By investigating various mechanisms implicated in MDP- and IFNγ-mediated signaling pathways, we revealed that the increased production of pro-inflammatory cytokines is highly dependent on the X-linked inhibitor of apoptosis protein (XIAP) but not on cellular IAP1 and IAP2. We also found that the NOD2 signaling pathway is regulated by the mammalian target of rapamycin (mTOR) but is not affected by phosphatidylinositol-3 kinase or signal transducer and activator of transcription 1 inhibition. Our results demonstrate, for the first time, that IFNγ positively affects NOD2-mediated signaling in human cDCs, in a manner considerably dependent on XIAP and partially dependent on mTOR.


Assuntos
Células Dendríticas/metabolismo , Interferon gama/farmacologia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Antígenos CD1/metabolismo , Citocinas/biossíntese , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Monócitos/citologia , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
7.
Cell Signal ; 28(5): 335-347, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26829212

RESUMO

BACKGROUND: BRAF-mutant melanoma is characterized by aggressive metastatic potential and therapeutic resistance. The innate immune receptor RIG-I has emerged as a potential target in melanoma therapies but the contributing pathways involved in anti-cancer activity are poorly characterized. METHODS: Baseline and ATRA-induced expression of RIG-I in nine (3 wild type and 6 BRAF-mutant) melanoma cell lines was measured with Q-PCR and Western blot. Ligand-specific stimulation of RIG-I was detected by Q-PCR and ELISA. Activation of the RIG-I-coupled IRF3, NF-κB and MAPK pathways was tested with protein array and Western blot. Cell proliferation and apoptosis was monitored by flow cytometry and cell counting. Down modulation of MKP-1 expression in melanoma cells was performed by specific siRNA. RESULTS: Short-term ATRA pre-treatment increases the expression of RIG-I in BRAF-mutant melanoma cells. Specific activation of RIG-I by 5'ppp-dsRNA leads to increased activity of the IRF3-IFNß pathway but does not influence NF-κB signaling. RIG-I mediates the targeted dephosphorylation of several MAPKs (p38, RSK1, GSK-3α/ß, HSP27) via the endogenous regulator MKP-1 resulting in decreased melanoma cell proliferation. CONCLUSION: RIG-I has the potential to exert anticancer activity in BRAF-mutant melanoma via controlling IFNß production and MAPK signaling. This is the first study showing that RIG-I activation results in MKP-1-mediated inhibition of cell proliferation via controlling the p38-HSP27, c-Jun and rpS6 pathways thus identifying RIG-I and MKP-1 as novel and promising therapeutical targets.


Assuntos
Proteína DEAD-box 58/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Sistema de Sinalização das MAP Quinases , Melanoma/enzimologia , Proteínas Proto-Oncogênicas B-raf/genética , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/metabolismo , Humanos , Fator Regulador 3 de Interferon/metabolismo , Melanoma/genética , Melanoma/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Tretinoína/farmacologia
8.
J Leukoc Biol ; 96(4): 579-89, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25001862

RESUMO

Type I and III IFNs are crucial, soluble components of potent antiviral responses. It has been explored recently that mTOR is involved in the regulation of IFN-α/ß production by pDCs, albeit its role in the induction of IFN responses in cDCs remained unrevealed. In this study, we demonstrate that the PI3K/mTOR pathway is indispensable for eliciting intact type I and III IFN responses in moDCs stimulated with polyI:C. The inhibition of mTOR functionality by rapamycin impairs the pIRF3 and also a few members of the MAPK family, suggesting that mTOR contributes to the activation of multiple signaling pathways in the presence of viral antigens. Furthermore, rapamycin-treated moDCs show decreased capacity to prime IFN-γ secretion by naive CD8(+) T-lymphocytes. As in moDCs, mTOR-mediated regulation is also essential for the production of type I and III IFNs in circulating CD1c(+) DCs. To our best knowledge, these results demonstrate for the first time that mTOR has an impact on the functional activities of cDCs via modulating the outcome of IFN secretion.


Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Antígenos CD1/metabolismo , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Células Dendríticas/efeitos dos fármacos , Expressão Gênica , Regulação da Expressão Gênica , Glicoproteínas/metabolismo , Humanos , Fator Regulador 3 de Interferon/genética , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Poli I-C/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Ativação Transcricional
9.
Appl Biochem Biotechnol ; 170(4): 819-30, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23613116

RESUMO

The glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily. Attachment of GITR to its ligand (GITRL) regulates diverse biological functions, including cell proliferation, differentiation, and survival. In this study, the extracellular region of human GITRL (hGITRL) was cloned, expressed, and purified. The coding sequence of the extracellular region of hGITRL was isolated from human brain cDNA and inserted in pET20b vector. The hGITRL was expressed in Escherichia coli BL21 (DE3) Star at 37 and 25 °C. The majority of the protein was found in inclusion bodies. We identified three important factors for efficient refolding of hGITRL: a ratio of GSH/GSSG, pH, and addition of polyethylene glycol. The renaturated protein was purified by Ni-NTA chromatography. The overall yield of the expression and refolding was higher than 50 mg/l E. coli culture grown at 37 °C. Size exclusion chromatography showed that hGITRL exists as mixture of various multimeric forms in solution. We tested the association of recombinant hGITRL with THP-1 and U937 cell lines and its activity to promote extracellular signal-regulated protein kinase phosphorylation. The results showed that the recombinant protein was biologically active.


Assuntos
Dobramento de Proteína , Multimerização Proteica , Fatores de Necrose Tumoral/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteína Relacionada a TNFR Induzida por Glucocorticoide/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Ligantes , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Necrose Tumoral/genética , Células U937
10.
Front Immunol ; 3: 207, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22848207

RESUMO

Cell death receptors have crucial roles in the regulation of immune responses. Here we review recent in vivo data confirming that the Fas death receptor (TNFSR6) on B cells is important for the regulation of autoimmunity since the impairment of only Fas function on B cells results in uncontrolled autoantibody production and autoimmunity. Fas plays a role in the elimination of the non-specific and autoreactive B cells in germinal center, while during the selection of antigen-specific B cells different escape signals ensure the resistance to Fas-mediated apoptosis. Antigen-specific survival such as BCR or MHCII signal or coreceptors (CD19) cooperating with BCR inhibits the formation of death inducing signaling complex. Antigen-specific survival can be reinforced by antigen-independent signals of IL-4 or CD40 overproducing the anti-apoptotic members of the Bcl-2 family proteins.

11.
J Immunol ; 189(6): 2815-23, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22891283

RESUMO

Activated T cells secrete Fas ligand (FasL)-containing vesicles (secreted vesicles) that induce death of target cells. We provide evidence that secreted vesicles from culture supernatants (Csup) of various origins are able to generate both Fas-dependent apoptotic and Fas-independent, nonapoptotic cell death. In the absence of Fas, the nonapoptotic, Fas-independent pathway could still induce cell death. In contrast to RIP-independent classical Fas-induced cell death triggered by cross-linked or membrane-bound FasL, CSup-derived stimuli-induced apoptosis exhibited unique molecular and enzymatic characteristics. It could be partially inhibited by blocking cathepsin D enzyme activity and required the presence of RIP. Whereas stimulation with CSup, derived from both FasL-overexpressing Jurkat cells and PBMC, could induce cell death, the requirements for Fas-associated death domain protein and caspase-9 were different between the two systems. Our study highlights an important distinction between cell contact-mediated and secreted vesicle-generated activation-induced cell death and also demonstrates that the type of the secreted vesicles can also modify the cell death route. We propose that besides cell-to-cell interaction-mediated Fas triggering, stimuli induced by secreted vesicles can mediate important additional cell death signals regulating activation-induced cell death under physiological conditions.


Assuntos
Apoptose/imunologia , Vesículas Citoplasmáticas/imunologia , Vesículas Citoplasmáticas/metabolismo , Ativação Linfocitária/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Receptor fas/fisiologia , Comunicação Celular/imunologia , Morte Celular/imunologia , Transformação Celular Neoplásica/imunologia , Técnicas de Cocultura , Meios de Cultivo Condicionados , Citidina Desaminase/fisiologia , Vesículas Citoplasmáticas/enzimologia , Testes Imunológicos de Citotoxicidade , Humanos , Células Jurkat , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Subpopulações de Linfócitos T/enzimologia
12.
Immunol Lett ; 143(1): 77-84, 2012 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-22553782

RESUMO

The death receptor, CD95/Fas, serves to eliminate potentially dangerous, self-reactive B cells. Engagement of B-cell receptors (BCR) on mature B-cells mediates the escape from cell death resulting in the activation and expansion of antigen specific clones. In addition to the antigen receptors, the receptors of B-cell activating factor belong to the tumor necrosis factor (TNF) family (BAFFR); moreover, the pattern recognition receptor, TLR9 may also deliver survival signals inhibiting Fas-mediated death of B-cells. Our aim was to compare the mechanism of BCR-induced and the BAFFR- or TLR9-stimulated rescue of B-cells from CD95/Fas-mediated apoptosis. We have found that BAFFR and TLR9 collaborate with BCR to protect B-cells from Fas-induced elimination and the rescue is independent of protein synthesis. The results revealed that the TLR9- and BCR-triggered rescue signals are transmitted through partially overlapping pathways; the protein kinase C (PKC) and the abl kinase induced phosphorylation may inactivate caspases in both CpG and anti-IgG stimulated cells. However, PI3-K activation is crucial upon the BCR driven anti-apoptotic effect, while p38 MAPK-mediated inactivation of caspases seems to play essential role in TLR9-mediated protection against Fas-induced programmed cell death.


Assuntos
Apoptose , Linfócitos B/imunologia , Caspases/metabolismo , Transdução de Sinais , Receptor Toll-Like 9/imunologia , Receptor fas/imunologia , Animais , Receptor do Fator Ativador de Células B/imunologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Camundongos , Receptores de Antígenos de Linfócitos B/imunologia , Receptor Toll-Like 9/metabolismo
13.
Cell Signal ; 21(2): 220-7, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18950707

RESUMO

B-cell fate during maturation and the germinal center reaction is regulated through the strength and the duration of the B-cell receptor signal. Signaling pathways discriminating between apoptosis and survival in B cells are keys in understanding adaptive immunity. Gab2 is a member of the Gab/Dos adaptor protein family. It has been shown in several model systems that Gab/Dos family members may regulate both the anti-apoptotic PI3-K/Akt and the mitogenic Ras/MAPK pathways, still their role in B-cells have not been investigated in detail. Here we studied the role of Gab2 in B-cell receptor mediated signaling. We have shown that BCR crosslinking induces the marked phosphorylation of Gab2 through both Lyn and Syk kinases. Subsequently Gab2 recruits p85 regulatory subunit of PI3-K, and SHP-2. Our results revealed that Ig-alpha/Ig-beta, signal transducing unit of the B-cell receptor, may function as scaffold recruiting Gab2 to the signalosome. Overexpression of Gab2 in A20 cells demonstrated that Gab2 is a regulator of the PI3-K/Akt but not that of the Ras/MAPK pathway in B cells. Accordingly to the elevated Akt phosphorylation, overexpression of wild-type Gab2 in A20 cells suppressed Fas-mediated apoptosis, and enhanced BCR-mediated rescue from Fas-induced cell death. Although PH-domain has only a stabilizing effect on membrane recruitment of Gab2, it is indispensable in mediating its anti-apoptotic effect.


Assuntos
Linfócitos B/imunologia , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Anti-Idiotípicos/farmacologia , Apoptose , Sítios de Ligação , Sobrevivência Celular , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/imunologia , Fosforilação , Transdução de Sinais , Receptor fas/imunologia , Receptor fas/metabolismo , Quinases da Família src/metabolismo
14.
Immunol Lett ; 116(2): 211-7, 2008 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-18243342

RESUMO

The survival of the mature resting B cells depends on signaling from B cell receptor (BCR), and a plethora of positive and negative regulators, that maintain cellular homeostasis and ultimately determine cell's fate, i.e., survival or programmed death (apoptosis). Among these regulators we have investigated the B cell activating factor belonging to tumor necrosis factor family (BAFF) and the prototypic death receptor Fas/CD95 mediated signals. We have shown that BAFF inhibits Fas-mediated cell death, however, the BCR-driven survival signals were not strengthened by BAFF. Therefore, we propose that BAFF may function independently of the antigen specificity of BCR, thus may enhance the risk of autoimmune diseases by promoting the survival of bystander B cells in the germinal center.


Assuntos
Fator Ativador de Células B/metabolismo , Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Receptor fas/metabolismo , Apoptose/imunologia , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Linfócitos B/imunologia , Linhagem Celular Tumoral , Humanos , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Receptor fas/genética
15.
Immunol Lett ; 92(1-2): 83-90, 2004 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-15081531

RESUMO

Type IIb Fcgamma receptors (FcgammaRIIb) have a major role in regulating B cell activation. Upon its co-aggregation with the B cell receptors (BCR) via immune complexes FcgammaRIIb become phosphorylated on tyrosine within its immunoreceptor tyrosine based inhibitory motif (ITIM) and in turn recruit protein- and inositol phosphatases, inhibiting thereby signal transduction. The intracellular domain of the human FcgammaRIIb has a membrane proximal motif that is very similar to those of MAPK docking site in MAPK-interacting molecules. Additionally, in contrast to the mouse, a serine residue is located next to this motif that is a potential phosphorylation site for Ser/Thr kinases. Our aim was to study the role of the putative MAPK docking motif on FcgammaRIIb mediated function. We report here that MAPKs bind to FcgammaRIIb affinity purified from the detergent extracts of anti-IgM activated and BCR-FcgammaRIIb co-clustered B cells. We detected extracellular signal regulated kinase (ERK) activity in FcgammaRIIb immunoprecipitates and identified the bound proteins as 85, 44 and 42kDa ERKs by Western blots. Active ERKs bound to the synthetic peptide representing the putative docking site of FcgammaRIIb on a Ser/Thr phosphatase dependent manner. The FcgammaRIIb-associated ERKs may phosphorylate the membrane proximal serine of the receptor. We examined the consequences of serine phosphorylation by comparing the proteins that interact with synthetic peptides comprising the combined sequences of the MAPK docking site and the ITIM either in phosphorylated or in non-phosphorylated forms. The results indicate that phosphorylation on serine modifies the binding of Lyn to FcgammaRIIb, thus might negatively regulate phosphorylation of ITIM.


Assuntos
Antígenos CD/imunologia , Proteínas Quinases Ativadas por Mitógeno/imunologia , Receptores de IgG/imunologia , Motivos de Aminoácidos , Antígenos CD/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fosforilação , Receptores de IgG/metabolismo , Serina/metabolismo
16.
Eur J Immunol ; 34(4): 1127-35, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15048724

RESUMO

Receptors specific for the Fc part of IgG (Fc gamma R) are expressed by several cell types and play diverse roles in immune responses. Impaired function of the activating and inhibitory Fc gamma R may result in autoimmunity. Thus, the modulation of IgG-Fc gamma R interaction can be a target for the development of treatments for some autoimmune and inflammatory diseases. This study addresses the localization and functional characterization of linear sequences in human IgG1 which bind to Fc gamma RII. Peptides with overlapping sequences derived from the CH2 domain of human IgG1 between P(234) and S(298) were synthesized and used in binding and functional experiments. Binding of the peptides to Fc gamma R was assayed in vitro and ex vivo, and peptides found to interact were functionally tested. The shortest effective peptide was T(256)-P(271), which bound to soluble recombinant Fc gamma RIIb with K(d)=6 x 10(6) M(-1). The biotinylated peptides R(255)-P(271) and T(256)-P(271) complexed by avidin exhibited functional activity; they induced Fc gamma RIIb-mediated inhibition of the BCR-triggered Ca(2+) response of human Burkitt lymphoma cells, and inflammatory cytokine production (TNF-alpha and IL-6) by the human monocyte cell line MonoMac. In conclusion, our results suggest that the selected peptides functionally represent the Fc gamma RII-binding part of IgG1.


Assuntos
Mapeamento de Epitopos , Imunoglobulina G/química , Monócitos/imunologia , Peptídeos/imunologia , Peptídeos/metabolismo , Receptores de IgG/química , Sítios de Ligação/imunologia , Western Blotting , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunoglobulina G/imunologia , Interleucina-6/metabolismo , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peptídeos/farmacologia , Fosforilação , Estrutura Quaternária de Proteína , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de IgG/genética , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
17.
Immunol Lett ; 82(1-2): 41-9, 2002 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-12008033

RESUMO

Ligation of B cell receptors (BCR) on immature B cells may induce apoptosis, while in mature B cells it stimulates cell activation and growth. The signaling pathway regulating the differential functional response, death or survival of the B cell is not fully characterized. We have tested the intracellular signaling requirement of these processes using B cells isolated from the spleen of irradiated auto-reconstituted (transitional immature B cells) and untreated mice (mature B cells), respectively. We compared the BCR induced intracellular [Ca2+] transient, protein tyrosine phosphorylation and ERK phosphorylation, furthermore, the activation of Elk-1 and CREB transcription factors. The BCR induced rise of intracellular [Ca2+] did not significantly differ in the two populations, only a slight difference in the late phase of the response was observed. Immature B cells responded with a maximum tyrosine phosphorylation to a five times lower dose of anti-IgM compared to the mature population. Most importantly, we have found a significant difference in the tyrosine phosphorylation of the Gab family adaptor proteins, Gab1/2. In contrast to mature B cells, crosslinking of BCR on immature B cells did not induce tyrosine phosphorylation of Gab2, thus the Gab2-organized signal amplification complex could not be produced. Furthermore, we detected a significant difference in the kinetics of BCR induced ERK, Elk-1 and CREB phosphorylation. In immature B cells, ERK was transiently phosphorylated, ceasing after 120 min, while in mature cells, ERK phosphorylation was sustained. Elk-1 and CREB activation was also transient in immature B cells, followed the kinetics of ERK phosphorylation. The lack of sustained Erk1/2 activation suppresses the transcription factors necessary for the proliferation signal. Since ERK is regulated by the phosphorylated Gab1/2, these data demonstrate that BCR triggered phosphorylation and signal amplification of Gab1/2 is a critical step in a life or death decision of B cells.


Assuntos
Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos de Diferenciação de Linfócitos B/análise , Apoptose , Subpopulações de Linfócitos B/classificação , Cálcio/metabolismo , Diferenciação Celular , Células Cultivadas , Cinética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fosfoproteínas/metabolismo , Fosforilação , Baço/citologia , Baço/imunologia , Células-Tronco/imunologia , Fatores de Transcrição/metabolismo
18.
Cell Signal ; 14(6): 563-72, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11897497

RESUMO

We have compared early signaling events at various stages of B cell differentiation using established mouse cell lines. Clustering of pre-B cell antigen receptor (BCR) or BCR induced the tyrosine phosphorylation of various proteins in all cells, although the phosphorylation pattern differed. In spite of the pre-BCR-induced tyrosine phosphorylation, we could not detect an intracellular Ca(2+) signal in pre-B cells. However, co-clustering of the pre-BCR with CD19 did induce Ca(2+) mobilization. In contrast to the immature and mature B cells, where the B cell linker protein (BLNK) went through inducible tyrosine phosphorylation upon BCR clustering, we observed a constitutive tyrosine phosphorylation of BLNK in pre-B cell lines. Both BLNK and phospholipase C (PLC)gamma were raft associated in unstimulated pre-B cells, and this could not be enhanced by pre-BCR engagement, suggesting a ligand-independent PLC gamma-mediated signaling. Further results indicate that the cell lines representing the immature stage are more sensitive to BCR-, CD19- and type II receptors binding the Fc part of IgG (Fc gamma RIIb)-mediated signals than mature B cells.


Assuntos
Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos CD/metabolismo , Antígenos CD19/metabolismo , Antígenos de Diferenciação de Linfócitos B/análise , Sinalização do Cálcio , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Diferenciação Celular , Linhagem Celular , Microdomínios da Membrana/química , Camundongos , Fosfatidilinositol 3-Quinases/fisiologia , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Agregação de Receptores , Receptores de IgG/metabolismo , Células-Tronco/metabolismo , Células Tumorais Cultivadas , Fosfolipases Tipo C/análise , Fosfolipases Tipo C/metabolismo
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