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1.
Eur J Immunol ; 49(12): 2172-2183, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31339552

RESUMO

Aggregation of IgE bound to high affinity IgE receptor (FcεRI) by multivalent antigen induces mast cell activation. Reportedly, disaggregation of aggregated FcεRI immediately terminated degranulation, and formation of co-ligated FcεRI and low affinity IgG receptor FcγRIIB blocked degranulation by inhibitory signal via SH2-containing inositol 5'-phosphatase 1 (SHIP1) phosphorylation. However, their molecular mechanisms to inhibit mast cell activation have been unclear in detail. Herein, we found that addition of excess monomeric hapten (TNP-alanine) to multivalent antigen (TNP-OVA)-activated rat basophilic leukemia cells and mouse bone marrow-derived mast cells induced immediate and transient Syk dephosphorylation, which was previously phosphorylated by TNP-OVA addition. Syk dephosphorylation correlated to rapidly decreased intracellular Ca2+ concentrations ([Ca2+ ]i ), terminated degranulation, and suppressed cytokine production through inhibition of Akt and ERK phosphorylation. Addition of hapten-specific IgG monoclonal antibody (anti-TNP IgG1) to activated mast cells induced translocation of SHIP1 to the plasma membrane and its phosphorylation, indicating that co-ligation of FcεRI and FcγRIIB after FcεRI aggregation can lead to SHIP1 activation. SHIP1 phosphorylation led to gradually decreased [Ca2+ ]i , weak inhibition of degranulation, and strong inhibition of cytokine production. Our findings clearly show the inhibitory mechanism of cell function in activated mast cells by operating Fc receptor crosslinking.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Haptenos/imunologia , Imunoglobulina G/imunologia , Mastócitos/imunologia , Animais , Linhagem Celular Tumoral , Capeamento Imunológico/imunologia , Mastócitos/citologia , Camundongos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/imunologia , Ratos , Receptores de IgE/imunologia , Receptores de IgG/imunologia
2.
J Autoimmun ; 53: 78-84, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24768065

RESUMO

The Mertk receptor tyrosine kinase facilitates macrophage and DC apoptotic-cell clearance and regulates immune tolerance. Mertk may also contribute to B-cell activation, because Mertk-KO mice fail to develop autoantibodies when allo-activated by T cells. We investigated this possibility with a well-characterized model in which injection of mice with goat anti-IgD antibody causes membrane IgD cross-linking that induces T-independent B cell activation and antigen presentation to T cells. Goat anti-mouse IgD antibody-injected C57BL/6 Mertk-KO mice had normal initial B cell activation and proliferation, but significantly lower T cell activation and proliferation, as well as lower IgE and IgG anti-goat IgG responses, as compared to C57BL/6 WT controls. B cell antigen processing, analyzed by evaluating B cell fluorescence following injection of monoclonal anti-IgD antibody labeled with biotin or FITC, was comparable between Mertk-KO mice and WT mice. IgD Ab primed B cells from Mertk-KO mice exhibited significantly lower ability in activating memory T cells isolated from WT mice injected with the same antigen 10 days before. These observations suggest that Mertk expression is required for optimal B-cell antigen presentation, which is, in turn, required in this model for optimal T cell activation and subsequent T cell-dependent B cell differentiation.


Assuntos
Linfócitos B/imunologia , Comunicação Celular/imunologia , Imunoglobulina D/imunologia , Proteínas Proto-Oncogênicas/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Linfócitos T/imunologia , Animais , Linfócitos B/citologia , Comunicação Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Imunoglobulina D/genética , Capeamento Imunológico/genética , Capeamento Imunológico/imunologia , Memória Imunológica/genética , Ativação Linfocitária/genética , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos B/genética , Linfócitos T/citologia , c-Mer Tirosina Quinase
3.
Eur J Immunol ; 44(6): 1802-13, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24610736

RESUMO

Along with MHC class I (MHCI), 2B4 provides nonredundant NK-cell inhibition in mice. The immunoregulatory role of 2B4 has been increasingly appreciated in models of tumor and viral infection, however, the interactions among 2B4, MHCI, and other activating NK-cell receptors remain uncertain. Here, we dissect the influence of two distinct inhibitory pathways in modulating NK-cell-mediated control of tumors expressing strong activating ligands, including RAE-1γ. In vitro cytotoxicity and in vivo peritoneal clearance assays using MHCI(+) CD48(+) (RMA-neo), MHCI(+) CD48(+) RAE-1γ (RMA-RAE-1γ), MHCI(-) CD48(+) (RMA-S-neo), and MHCI(-) CD48(+) RAE-1γ (RMA-S-RAE-1γ) tumor lines demonstrated that NKG2D activation supersedes the inhibitory effect of both 2B4- and MHCI-mediated immune-tolerance systems. Furthermore, 2B4KO mice subcutaneously challenged with RMA-neo and RMA-S-neo exhibited reduced tumor growth and significantly prolonged survival compared with WT mice, implying that 2B4 is constitutively engaged in the NK-cell tolerance mechanism in vivo. Nevertheless, the inhibitory effect of 2B4 is significantly attenuated when NK cells encountered highly stressed tumor cells expressing RAE-1γ, resulting in an immune response shift toward NK-cell activation and tumor regression. Therefore, our data highlight the importance of the 2B4-mediated inhibitory system as an alternate self-tolerance mechanism, whose role can be modulated by the strength of activating receptor signaling within the tumor microenvironment.


Assuntos
Antígenos CD/imunologia , Capeamento Imunológico/imunologia , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptores Imunológicos/imunologia , Tolerância a Antígenos Próprios , Animais , Antígenos CD/genética , Linhagem Celular Tumoral , Feminino , Capeamento Imunológico/genética , Células Matadoras Naturais/patologia , Camundongos , Camundongos Knockout , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Receptores Imunológicos/genética , Família de Moléculas de Sinalização da Ativação Linfocitária , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
4.
J Biol Chem ; 286(48): 41852-41861, 2011 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-21994939

RESUMO

Ubiquitination has been implicated in negatively regulating insulin-like growth factor I receptor (IGF-IR) activity. Because of the relative stability of IGF-IR in the presence of ligand stimulation, IGF-IR ubiquitination sites have yet to be mapped and characterized, thus preventing a direct demonstration of how the receptor ubiquitination contributes to downstream molecular cascades. We took advantage of an anti-IGF-IR antibody (h10H5) that induces more efficient receptor down-regulation to show that IGF-IR is promptly and robustly ubiquitinated. The ubiquitination sites were mapped to the two lysine residues in the IGF-IR activation loop (Lys-1138 and Lys-1141) and consisted of polyubiquitin chains formed through both Lys-48 and Lys-29 linkages. Mutation of these ubiquitinated lysine residues resulted in decreased h10H5-induced IGF-IR internalization and down-regulation as well as a reduced cellular response to h10H5 treatment. We have therefore demonstrated that IGF-IR ubiquitination contributes critically to the down-regulating and antiproliferative activity of h10H5. This finding is physiologically relevant because insulin-like growth factor I appears to mediate ubiquitination of the same major sites as h10H5 (albeit to a lesser extent), and ubiquitination is facilitated by pre-existing phosphorylation of the receptor in both cases. Furthermore, identification of a breast cancer cell line with a defect in IGF-IR ubiquitination suggests that this could be an important tumor resistance mechanism to evade down-regulation-mediated negative regulation of IGF-IR activity in cancer.


Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Capeamento Imunológico/efeitos dos fármacos , Receptor IGF Tipo 1/metabolismo , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais Murinos/imunologia , Linhagem Celular Tumoral , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Humanos , Capeamento Imunológico/genética , Capeamento Imunológico/imunologia , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/imunologia , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/imunologia , Ubiquitinação
5.
J Immunol ; 187(4): 1754-63, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21742972

RESUMO

A high activatory/inhibitory FcγR binding ratio is critical for the activity of mAb such as rituximab and alemtuzumab that attack cancer cells directly and eliminate them by recruiting immune effectors. Optimal FcγR binding profiles of other anti-cancer mAb, such as immunostimulatory mAb that stimulate or block immune receptors, are less clear. In this study, we analyzed the importance of isotype and FcγR interactions in controlling the agonistic activity of the anti-mouse CD40 mAb 3/23. Mouse IgG1 (m1) and IgG2a (m2a) variants of the parental 3/23 (rat IgG2a) were engineered and used to promote humoral and cellular responses against OVA. The mouse IgG1 3/23 was highly agonistic and outperformed the parental Ab when promoting Ab (10-100-fold) and T cell (OTI and OTII) responses (2- to >10-fold). In contrast, m2a was almost completely inactive. Studies in FcγR knockout mice demonstrated a critical role for the inhibitory FcγRIIB in 3/23 activity, whereas activatory FcγR (FcγRI, -III, and -IV) was dispensable. In vitro experiments established that the stimulatory effect of FcγRIIB was mediated through Ab cross-linking delivered in trans between neighboring cells and did not require intracellular signaling. Intriguingly, activatory FcγR provided effective cross-linking of 3/23 m2a in vitro, suggesting the critical role of FcγRIIB in vivo reflects its cellular distribution and bioavailability as much as its affinity for a particular Ab isotype. In conclusion, we demonstrate an essential cross-linking role for the inhibitory FcγRIIB in anti-CD40 immunostimulatory activity and suggest that isotype will be an important issue when optimizing reagents for clinical use.


Assuntos
Anticorpos Monoclonais Murinos , Antineoplásicos , Antígenos CD40/imunologia , Capeamento Imunológico/efeitos dos fármacos , Receptores de IgG/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacocinética , Anticorpos Monoclonais Murinos/farmacologia , Antineoplásicos/imunologia , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Capeamento Imunológico/imunologia , Camundongos , Camundongos Knockout , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia
6.
Blood ; 117(23): 6143-51, 2011 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-21357766

RESUMO

The inhibition of vaccination by maternal antibodies is a widely observed phenomenon in human and veterinary medicine. Maternal antibodies are known to suppress the B-cell response. This is similar to antibody feedback mechanism studies where passively transferred antibody inhibits the B-cell response against particulate antigens because of epitope masking. In the absence of experimental data addressing the mechanism underlying inhibition by maternal antibodies, it has been suggested that epitope masking explains the inhibition by maternal antibodies, too. Here we report that in the cotton rat model of measles virus (MV) vaccination passively transferred MV-specific immunoglobulin G inhibit B-cell responses through cross-linking of the B-cell receptor with FcγRIIB. The extent of inhibition increases with the number of antibodies engaging FcγRIIB and depends on the Fc region of antibody and its isotype. This inhibition can be partially overcome by injection of MV-specific monoclonal IgM antibody. IgM stimulates the B-cell directly through cross-linking the B-cell receptor via complement protein 3d and antigen to the complement receptor 2 signaling complex. These data demonstrate that maternal antibodies inhibit B-cell responses by interaction with the inhibitory/regulatory FcγRIIB receptor and not through epitope masking.


Assuntos
Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Imunoglobulina G/imunologia , Troca Materno-Fetal/imunologia , Vacina contra Sarampo/farmacologia , Vacinação , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Complemento C3d/imunologia , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina M/imunologia , Imunoglobulina M/farmacologia , Capeamento Imunológico/efeitos dos fármacos , Capeamento Imunológico/imunologia , Troca Materno-Fetal/efeitos dos fármacos , Gravidez , Receptores de Complemento 3d/imunologia , Receptores de IgG/imunologia , Sigmodontinae
7.
Immunity ; 32(2): 187-99, 2010 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-20171124

RESUMO

Early events of B cell activation after B cell receptor (BCR) triggering have been well characterized. However, little is known about the steady state of the BCR on the cell surface. Here, we simultaneously visualize single BCR particles and components of the membrane skeleton. We show that an ezrin- and actin-defined network influenced steady-state BCR diffusion by creating boundaries that restrict BCR diffusion. We identified the intracellular domain of Igbeta as important in mediating this restriction in diffusion. Importantly, alteration of this network was sufficient to induce robust intracellular signaling and concomitant increase in BCR mobility. Moreover, by using B cells deficient in key signaling molecules, we show that this signaling was most probably initiated by the BCR. Thus, our results suggest the membrane skeleton plays a crucial function in controlling BCR dynamics and thereby signaling, in a way that could be important for understanding tonic signaling necessary for B cell development and survival.


Assuntos
Actinas/metabolismo , Linfócitos B/metabolismo , Antígenos CD79/metabolismo , Membrana Celular/imunologia , Proteínas do Citoesqueleto/metabolismo , Actinas/imunologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Antígenos CD79/genética , Antígenos CD79/imunologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/imunologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/imunologia , Capeamento Imunológico/efeitos dos fármacos , Capeamento Imunológico/genética , Capeamento Imunológico/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Ligação Proteica , Engenharia de Proteínas , Estrutura Terciária de Proteína/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Tiazolidinas/farmacologia
8.
J Leukoc Biol ; 86(6): 1351-8, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19741159

RESUMO

Activation of the high-affinity receptor for IgE, FcepsilonRI, is known to elicit its rapid down-regulation through internalization and degradation. In keeping with this, expression of all three FcepsilonRI subunits is decreased at the protein level after cross-linkage of IgE with antigen. However, we find that the FcepsilonRI beta-subunit is also selectively suppressed at the mRNA level, through a pathway primarily involving Fyn, Syk, PI3K, and NF-kappaB. IgG or calcium ionophore, stimuli known to mimic portions of the IgE signaling cascade, similarly suppressed beta-subunit expression. LPS, a NF-kappaB-activating TLR ligand, did not alter beta-subunit expression. As IgE increases FcepsilonRI expression, we examined the coordinated regulation of FcepsilonRI subunits during culture with IgE, followed by cross-linkage with antigen. IgE increased the expression of all three FcepsilonRI subunits and strikingly induced expression of the antagonistic beta(T). The ratio of beta:beta(T) protein expression decreased significantly during culture with IgE and was reset to starting levels by antigen cross-linkage. These changes in protein levels were matched by similar fluctuations in beta and beta(T) mRNAs. FcepsilonRIbeta is a key regulator of IgER expression and function, a gene in which polymorphisms correlate with allergic disease prevalence. The ability of IgE and FcepsilonRI signaling to coordinate expression of the beta and beta(T) subunits may comprise a homeostatic feedback loop-one that could promote chronic inflammation and allergic disease if dysregulated.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunoglobulina E/imunologia , Capeamento Imunológico/imunologia , Receptores de IgE/imunologia , Transdução de Sinais/imunologia , Animais , Regulação da Expressão Gênica/genética , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Imunoglobulina E/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Capeamento Imunológico/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/imunologia , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Polimorfismo Genético/genética , Polimorfismo Genético/imunologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/imunologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Receptores de IgE/biossíntese , Receptores de IgE/genética , Transdução de Sinais/genética , Quinase Syk
9.
Results Probl Cell Differ ; 49: 63-85, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19132322

RESUMO

Internalization of cell surface receptors has long been regarded as a pure means to terminate signaling via receptor degradation. A growing body of information points to the fact that many internalized receptors are still in their active state and that signaling continues along the endocytic pathway. Thus endocytosis orchestrates cell signaling by coupling and integrating different cascades on the surface of endocytic vesicles to control the quality, duration, intensity, and distribution of signaling events. The death receptors tumor necrosis factor-receptor 1 (TNF-R1) and CD95 (Fas, APO-1) are known not only to signal for cell death via apoptosis but are also capable of inducing antiapoptotic signals via transcription factor NF-kappaB induction or activation of the proliferative mitogen-activated protein kinase (MAPK)/ERK (extracellular signal-regulated kinase) protein kinase cascades, resulting in cell protection and tissue regeneration. A clue to the understanding of these contradictory biological phenomena may arise from recent findings which reveal a regulatory role of receptor internalization and intracellular receptor trafficking in selectively transmitting signals, which lead either to apoptosis or to the survival of the cell. In this chapter, we discuss the dichotomy of pro- and antiapoptotic signaling of the death receptors TNF-R1 and CD95. First, we will address the role of lipid rafts and post-translational modifications of death receptors in regulating the formation of receptor complexes. Then, we will discuss the role of internalization in determining the fate of the receptors and subsequently the specificity of signaling events. We propose that fusion of internalized TNF-receptosomes with trans-Golgi vesicles should be recognized as a novel mechanism to transduce death signals along the endocytic route. Finally, the lessons learnt from the strategy of adenovirus to escape apoptosis by targeting death receptor internalization demonstrate the biological significance of TNF receptor compartmentalization for immunosurveillance.


Assuntos
Apoptose/imunologia , Endocitose/imunologia , Capeamento Imunológico/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Receptor fas/imunologia , Animais , Humanos
10.
Mol Immunol ; 46(5): 793-802, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18945491

RESUMO

In addition to initiating signaling cascades leading to mast cell mediator release, aggregation of the high affinity IgE receptor (FcvarepsilonRI) leads to rapid internalization of the cross-linked receptor. However, little is known about the trafficking of the internalized FcvarepsilonRI. Here we demonstrate that in RBL-2H3 cells, aggregated FcvarepsilonRI appears in the early endosomal antigen 1 (EEA1(+)) domains of the early endosomes within 15min after ligation. Minimal co-localization of FcvarepsilonRI with Rab5 was observed by 30min, followed by its appearance in the Rab7(+) late endosomes and lysosomes at later time points. During endosomal sorting, FcvarepsilonRIalpha and gamma subunits remain associated. In Syk-deficient RBL-2H3 cells, the rate of transport to lysosomes is markedly increased. Taken together, our data demonstrate time-dependent sorting of aggregated FcvarepsilonRI within the endosomal-lysosomal network, and that Syk may play an essential role in regulating the trafficking and retention of FcvarepsilonRI in endosomes.


Assuntos
Endossomos/imunologia , Capeamento Imunológico/imunologia , Lisossomos/imunologia , Receptores de IgE/imunologia , Animais , Linhagem Celular , Endossomos/genética , Capeamento Imunológico/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Lisossomos/genética , Transporte Proteico/genética , Transporte Proteico/imunologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Ratos , Receptores de IgE/genética , Quinase Syk , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/imunologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/imunologia , proteínas de unión al GTP Rab7
11.
Blood ; 113(16): 3706-15, 2009 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-19023113

RESUMO

Subjects with X-linked hyper-IgM syndrome (X-HIgM) have a markedly reduced frequency of CD27(+) memory B cells, and their Ig genes have a low level of somatic hypermutation (SHM). To analyze the nature of SHM in X-HIgM, we sequenced 209 nonproductive and 926 productive Ig heavy chain genes. In nonproductive rearrangements that were not subjected to selection, as well as productive rearrangements, most of the mutations were within targeted RGYW, WRCY, WA, or TW motifs (R = purine, Y = pyrimidine, and W = A or T). However, there was significantly decreased targeting of the hypermutable G in RGYW motifs. Moreover, the ratio of transitions to transversions was markedly increased compared with normal. Microarray analysis documented that specific genes involved in SHM, including activation-induced cytidine deaminase (AICDA) and uracil-DNA glycosylase (UNG2), were up-regulated in normal germinal center (GC) B cells, but not induced by CD40 ligation. Similar results were obtained from light chain rearrangements. These results indicate that in the absence of CD40-CD154 interactions, there is a marked reduction in SHM and, specifically, mutations of AICDA-targeted G residues in RGYW motifs along with a decrease in transversions normally related to UNG2 activity.


Assuntos
Linfócitos B/enzimologia , Citidina Desaminase/biossíntese , DNA Glicosilases/biossíntese , Regulação Enzimológica da Expressão Gênica/genética , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/genética , Cadeias Pesadas de Imunoglobulinas/genética , Hipermutação Somática de Imunoglobulina/genética , Adolescente , Adulto , Linfócitos B/imunologia , Antígenos CD40/genética , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Ligante de CD40/genética , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Criança , Citidina Desaminase/genética , Citidina Desaminase/imunologia , DNA Glicosilases/genética , DNA Glicosilases/imunologia , Análise Mutacional de DNA , Regulação Enzimológica da Expressão Gênica/imunologia , Centro Germinativo/enzimologia , Centro Germinativo/imunologia , Humanos , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/enzimologia , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Capeamento Imunológico/genética , Capeamento Imunológico/imunologia , Memória Imunológica/genética , Masculino , Mutação , Hipermutação Somática de Imunoglobulina/imunologia , Regulação para Cima/genética , Regulação para Cima/imunologia
12.
Blood ; 113(2): 358-69, 2009 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-18922855

RESUMO

NKp80, an activating homodimeric C-type lectin-like receptor (CTLR), is expressed on essentially all human natural killer (NK) cells and stimulates their cytotoxicity and cytokine release. Recently, we demonstrated that the ligand for NKp80 is the myeloid-specific CTLR activation-induced C-type lectin (AICL), which is encoded in the natural killer gene complex (NKC) adjacent to NKp80. Here, we show that NKp80 also is expressed on a minor fraction of human CD8 T cells that exhibit a high responsiveness and an effector memory phenotype. Gene expression profiling and flow cytometric analyses revealed that this NKp80(+) T-cell subset is characterized by the coexpression of other NK receptors and increased levels of cytotoxic effector molecules and adhesion molecules mediating access to sites of inflammation. NKp80 ligation augmented CD3-stimulated degranulation and interferon (IFN)gamma secretion by effector memory T cells. Furthermore, engagement of NKp80 by AICL-expressing transfectants or macrophages markedly enhanced CD8 T-cell responses in alloreactive settings. Collectively, our data demonstrate that NKp80 is expressed on a highly responsive subset of effector memory CD8 T cells with an inflammatory NK-like phenotype and promotes T-cell responses toward AICL-expressing cells. Hence, NKp80 may enable effector memory CD8 T cells to interact functionally with cells of myeloid origin at sites of inflammation.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Regulação da Expressão Gênica/imunologia , Lectinas Tipo C/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T CD8-Positivos/metabolismo , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Linhagem Celular , Perfilação da Expressão Gênica/métodos , Humanos , Capeamento Imunológico/genética , Capeamento Imunológico/imunologia , Memória Imunológica/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Lectinas Tipo C/biossíntese , Lectinas Tipo C/genética , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Células Mieloides/imunologia , Células Mieloides/metabolismo , Receptores de Células Matadoras Naturais/biossíntese , Receptores de Células Matadoras Naturais/genética , Subpopulações de Linfócitos T/metabolismo
13.
J Immunol ; 181(11): 7863-72, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19017976

RESUMO

Myeloid dendritic cells (mDC) activated with a B7-DC-specific cross-linking IgM Ab (B7-DC XAb) take up and retain Ag and interact with T cell compartments to affect a number of biologic changes that together cause strong antitumor responses and blockade of inflammatory airway disease in animal models. The molecular events mediating the initial responses in mDC remain unclear. In this study we show that B7-DC XAb caused rapid phosphorylation of the adaptor protein DAP12 and intracellular kinases Syk and phospholipase C-gamma1. Pretreatment of mDC with the Syk inhibitor piceatannol blocked B7-DC XAb-induced Ag uptake with a concomitant loss of tumor protection in mice. Vaccination with tumor lysate-pulsed wild-type B7-DC XAb-activated mDC, but not TREM-2 knockout XAb-activated mDC, protected mice from lethal melanoma challenge. Multimolecular caps appeared within minutes of B7-DC XAb binding to either human or mouse mDC, and FRET analysis showed that class II, CD80, CD86, and TREM-2 are recruited in tight association on the cell surface. When TREM-2 expression was reduced in wild-type mDC using short hairpin RNA or by using mDC from TREM-2 knockout mice, in vitro DC failed to take up Ag after B7-DC XAb stimulation. These results directly link TREM-2 signaling with one change in the mDC phenotype that occurs in response to this unique Ab. The parallel signaling events observed in both human and mouse mDC support the hypothesis that B7-DC cross-linking may be useful as a therapeutic immune modulator in human patients.


Assuntos
Antígenos/imunologia , Células Dendríticas/imunologia , Glicoproteínas de Membrana/imunologia , Células Mieloides/imunologia , Receptores Imunológicos/imunologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Anticorpos/farmacologia , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Capeamento Imunológico/efeitos dos fármacos , Capeamento Imunológico/genética , Capeamento Imunológico/imunologia , Inflamação/genética , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Melanoma/genética , Melanoma/imunologia , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Fosfolipase C gama/genética , Fosfolipase C gama/imunologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Fosforilação/imunologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/genética , Transtornos Respiratórios/genética , Transtornos Respiratórios/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Estilbenos/farmacologia , Quinase Syk , Linfócitos T/imunologia
14.
J Immunol ; 181(8): 5501-9, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18832707

RESUMO

Phagocytosis of IgG-opsonized microbes via the Fc gamma receptor (Fc gammaR) requires the precise coordination of a number of signaling molecules, including the low-molecular mass GTPases. Little is known about the Ras-family GTPase Rap1 in this process. We therefore investigated its importance in mediating Fc gammaR-dependent phagocytosis in NR8383 rat alveolar macrophages. Pulldown of active Rap1 and fluorescence microscopic analysis of GFP-RalGDS (Ral guanine dissociation stimulator)-transfected macrophages revealed that Rap1 is indeed activated by Fc gammaR crosslinking. Inhibition of Rap1 activity, both by Rap1GAP (GTPase-activating protein) expression and liposome-delivered blocking Ab, severely impaired the ability of cells to ingest IgG-opsonized targets. Fc gammaR-induced Rap1 activation was found to be independent of both cAMP and Ca(2+), suggesting a role for the second messenger-independent guanosine exchange factor, C3G. This was supported by the facts that 1) liposome-delivered blocking Ab against C3G inhibited both Fc gammaR-dependent phagocytosis and Rap1 activation, and 2) both active Rap1GTP and C3G were found to translocate to the phagosome. Taken together, our data demonstrate a novel role for Rap1 and its exchange factor C3G in mediating Fc gammaR-dependent phagocytosis.


Assuntos
Macrófagos Alveolares/imunologia , Fagocitose/imunologia , Receptores de IgG/imunologia , Proteínas rap1 de Ligação ao GTP/imunologia , Animais , Cálcio/imunologia , AMP Cíclico/imunologia , Fator 2 de Liberação do Nucleotídeo Guanina/imunologia , Humanos , Capeamento Imunológico/efeitos dos fármacos , Capeamento Imunológico/imunologia , Lipossomos , Fagocitose/efeitos dos fármacos , Ratos , Sistemas do Segundo Mensageiro/imunologia , Células U937
15.
J Immunol ; 180(9): 5916-26, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18424711

RESUMO

Lymphocyte activation gene-3 (LAG-3) is a CD4-related transmembrane protein expressed by regulatory T cells that binds MHC II on APCs. It is shown in this study that during Treg:DC interactions, LAG-3 engagement with MHC class II inhibits DC activation. MHC II cross-linking by agonistic Abs induces an ITAM-mediated inhibitory signaling pathway, involving FcgammaRgamma and ERK-mediated recruitment of SHP-1 that suppresses dendritic cell maturation and immunostimulatory capacity. These data reveal a novel ITAM-mediated inhibitory signaling pathway in DCs triggered by MHC II engagement of LAG-3, providing a molecular mechanism in which regulatory T cells may suppress via modulating DC function.


Assuntos
Antígenos CD/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Anticorpos/farmacologia , Antígenos CD/metabolismo , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Capeamento Imunológico/efeitos dos fármacos , Capeamento Imunológico/imunologia , Camundongos , Camundongos Knockout , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Proteína do Gene 3 de Ativação de Linfócitos
16.
J Immunol ; 180(9): 6064-9, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18424727

RESUMO

Through its receptor Kit (CD117), stem cell factor (SCF) critically regulates human mast cell (MC) differentiation, survival, priming, and activation. The dominance of SCF in setting these parameters compels stringent contra-regulation to maintain a balanced MC phenotype. We have synthesized a library of bispecific Ab fragments to examine the effect of linking Kit with CD300a. In this study, we report that CD300a exerts a strong inhibitory effect on Kit-mediated SCF-induced signaling, consequently impairing MC differentiation, survival, and activation in vitro. This effect derives from Kit-mediated tyrosine phosphorylation of CD300a and recruitment of the SHIP-1 but not of SH2-containing protein phosphatase 1. CD300a inhibits the constitutive activation of the human leukemic HMC-1 cells but not their survival. Finally, CD300a abrogates the allergic reaction induced by SCF in a murine model of cutaneous anaphylaxis. Our findings highlight CD300a as a novel regulator of Kit in human MC and suggest roles for this receptor as a suppressor of Kit signaling in MC-related disorders.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antígenos CD/imunologia , Capeamento Imunológico/efeitos dos fármacos , Mastócitos/imunologia , Proteínas Proto-Oncogênicas c-kit/imunologia , Receptores Imunológicos/imunologia , Transdução de Sinais/efeitos dos fármacos , Anafilaxia/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Modelos Animais de Doenças , Humanos , Capeamento Imunológico/imunologia , Camundongos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/imunologia , Fator de Células-Tronco/imunologia
17.
Blood ; 111(7): 3562-70, 2008 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-18198349

RESUMO

Data from several investigators suggest that the alpha2beta1 integrin, a receptor for collagens, laminins, decorin, E-cadherin, matrix metalloproteinase-1, endorepellin, and several viruses, is required for innate immunity and regulation of autoimmune/allergic disorders. We demonstrated that the innate immune response to Listeria monocytogenes required alpha2beta1 integrin expression by peritoneal mast cells (PMCs). Ligation of the alpha2beta1 integrin by C1q contained in immune complexes comprised of Listeria and antibody was required for PMC activation in vitro and in vivo. However, ligation of the alpha2beta1 integrin alone was insufficient to activate cytokine secretion, suggesting that one or more additional signals emanating from a coreceptor were required for PMC activation. Here, we demonstrate that C1q, but neither other complement proteins nor FcRgamma, is required for early innate immune response to Listeria. The binding of Listeria's Internalin B (InlB) to hepatocyte growth factor receptor (HGF-R)/c-met provides the costimulatory function required for PMC activation. Either HGF or Listeria InlB bound to c-met and either C1q or type I collagen bound to alpha2beta1 integrin stimulates PMC activation. These findings suggest that crosstalk between c-met and the alpha2beta1 integrin may contribute to mast-cell activation in autoimmune and inflammatory disorders.


Assuntos
Imunidade Inata/fisiologia , Integrina alfa2/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Mastócitos/imunologia , Proteínas Proto-Oncogênicas c-met/imunologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Complemento C1q/genética , Complemento C1q/imunologia , Citocinas/genética , Citocinas/imunologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/imunologia , Capeamento Imunológico/imunologia , Inflamação/genética , Inflamação/imunologia , Integrina alfa2/genética , Leucócitos Mononucleares/imunologia , Listeriose/genética , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Ligação Proteica/imunologia , Proteínas Proto-Oncogênicas c-met/genética , Receptores de IgG/genética , Receptores de IgG/imunologia , Receptores Virais/genética , Receptores Virais/imunologia
18.
Blood ; 111(7): 3653-64, 2008 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-18212246

RESUMO

During immunologic synapse (IS) formation, human CD38 redistributes to the contact area of T cell-antigen-presenting cell (APC) conjugates in an antigen-dependent manner. Confocal microscopy showed that CD38 preferentially accumulated along the contact zone, whereas CD3-zeta redistributed toward the central zone of the IS. APC conjugates with human T cells or B cells transiently expressing CD38-green fluorescent protein revealed the presence of 2 distinct pools of CD38, one localized at the cell membrane and the other in recycling endosomes. Both pools were recruited to the T/APC contact sites and required antigen-pulsed APCs. The process appeared more efficient in T cells than in APCs. CD38 was actively recruited at the IS of T cells by means of Lck-mediated signals. Overexpression of CD38 in T cells increased the levels of antigen-induced intracellular calcium release. Opposite results were obtained by down-regulating surface CD38 expression by means of CD38 siRNA. CD38 blockade in influenza HA-specific T cells inhibited IL-2 and IFN-gamma production, PKC phosphorylation at Thr538, and PKC recruitment to the IS induced by antigen-pulsed APCs. These results reveal a new role for CD38 in modulating antigen-mediated T-cell responses during IS formation.


Assuntos
ADP-Ribosil Ciclase 1/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígenos Virais/imunologia , Sinalização do Cálcio/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Capeamento Imunológico/imunologia , Glicoproteínas de Membrana/imunologia , Linfócitos T/imunologia , ADP-Ribosil Ciclase 1/antagonistas & inibidores , ADP-Ribosil Ciclase 1/genética , Células Apresentadoras de Antígenos/citologia , Complexo CD3/genética , Complexo CD3/imunologia , Sinalização do Cálcio/genética , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Endossomos/genética , Endossomos/imunologia , Humanos , Capeamento Imunológico/genética , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Linfócitos T/citologia
19.
Blood ; 111(7): 3644-52, 2008 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-18223169

RESUMO

CD38 is a surface receptor able to induce activation, proliferation, and survival of human and mouse lymphocytes; this molecule is expressed on the surface of both mature and immature B cells. In this work, the function of CD38 in the maturation of murine B lymphocytes in the spleen was analyzed. The results showed that CD38 is highly expressed on Transitional 2 (T2) B lymphocytes with an intermediate expression on Transitional 1 (T1) and mature follicular B cells (M). Correlating with a high expression of CD38, T2 cells are also larger and more granular than T1 or M B cells. T2 cells also showed high levels of other molecules, which indicate an activated phenotype. CD38 crosslinking induced proliferation and maturation of T2 B lymphocytes; in contrast, T1 subset died by apoptosis. Finally, CD38 stimulation of T2 B lymphocytes obtained from Btk-, Lyn-, or Fyn-deficient mice showed a defective differentiation; similarly, drugs interfering with PI3K or ERK decreased the proliferation or differentiation of this subset. This suggests that these molecules participate in the CD38 signaling pathway. As a whole, the results indicate that CD38 plays an important role in the regulation of B-cell maturation in the spleen.


Assuntos
ADP-Ribosil Ciclase 1/imunologia , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Glicoproteínas de Membrana/imunologia , Transdução de Sinais/imunologia , Baço/imunologia , ADP-Ribosil Ciclase 1/genética , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/citologia , Diferenciação Celular/genética , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Regulação da Expressão Gênica/genética , Humanos , Capeamento Imunológico/genética , Capeamento Imunológico/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/imunologia , Transdução de Sinais/genética , Baço/citologia , Quinases da Família src/genética , Quinases da Família src/imunologia
20.
Blood ; 111(2): 517-24, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17938255

RESUMO

The clonal expansion of chronic lymphocytic leukemia (CLL) cells requires the interaction with the microenvironment and is under the control of several cytokines. Here, we investigated the effect of IL-15 and IL-21, which are closely related to IL-2 and share the usage of the common gamma chain and of its JAK3-associated pathway. We found remarkable differences in the signal transduction pathways activated by these cytokines, which determined different responses in CLL cells. IL-15 caused cell proliferation and prevented apoptosis induced by surface IgM cross-linking. These effects were more evident in cells stimulated via surface CD40, which exhibited increased cell expression of IL-15Ralpha chain and, in some of the cases, also of IL-2Rbeta. IL-21 failed to induce CLL cell proliferation and instead promoted apoptosis. Following cell exposure to IL-15, phosphorylation of STAT5 was predominantly observed, whereas, following stimulation with IL-21, there was predominant STAT1 and STAT3 activation. Moreover, IL-15 but not IL-21 caused an increased phosphorylation of Shc and ERK1/2. Pharmacological inhibition of JAK3 or of MEK, which phosphorylates ERK1/2, efficiently blocked IL-15-induced CLL cell proliferation and the antiapoptotic effect of this cytokine. The knowledge of the signaling pathways regulating CLL cell survival and proliferation may provide new molecular targets for therapeutic intervention.


Assuntos
Interleucina-15/imunologia , Interleucinas/imunologia , Janus Quinase 3/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Fatores de Transcrição STAT/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Humanos , Imunoglobulina M/imunologia , Capeamento Imunológico/efeitos dos fármacos , Capeamento Imunológico/imunologia , Interleucina-15/farmacologia , Interleucina-2/imunologia , Subunidade beta de Receptor de Interleucina-2/imunologia , Interleucinas/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/terapia , MAP Quinase Quinase Quinases/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacos , Receptores de Interleucina-15/imunologia , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src
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