RESUMO
A therapeutic platform-drug-free macromolecular therapeutics (DFMT)-that induces apoptosis in B cells by cross-linking of CD20 receptors, without the need for low molecular weight cytotoxic drug, is developed. In this report, a DFMT system is synthesized and evaluated based on human serum albumin (HSA) and two complementary coiled-coil forming peptides, CCE and CCK. Fab' fragment of anti-CD20 monoclonal antibody rituximab is attached to CCE (Fab'-CCE); multiple grafts of CCK are conjugated to HSA (HSA-(CCK)7 ). The colocalization of both nanoconjugates at the surface of non-Hodgkin's lymphoma (NHL) Raji cells is demonstrated by confocal fluorescence microscopy. The colocalization leads to coiled-coil formation, CD20 cross-linking, and apoptosis induction. The apoptotic levels are evaluated by Annexin V, Caspase 3, and terminal deoxynucleotidyl transferase dUTP nick end labeling assays. Selective surface binding of DFMT to CD20+ cells is validated in experiments on a coculture of CD20+ (Raji) and CD20-(DG-75) cells. It is found that DFMT can trigger calcium influx only in Raji cells, but not in DG-75 cells. A highly specific treatment for NHL and other B cell malignancies with considerable translational potential is presented by HSA-based DFMT system.
Assuntos
Apoptose/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas , Capeamento Imunológico/efeitos dos fármacos , Linfoma de Células B/tratamento farmacológico , Peptídeos , Rituximab , Albumina Sérica Humana , Linhagem Celular Tumoral , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/farmacologia , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Peptídeos/química , Peptídeos/farmacologia , Rituximab/química , Rituximab/farmacologia , Albumina Sérica Humana/química , Albumina Sérica Humana/farmacologiaRESUMO
Epratuzumab has demonstrated therapeutic activity in patients with non-Hodgkin lymphoma, acute lymphoblastic leukemia, systemic lupus erythematosus, and Sjögren's syndrome, but its mechanism of affecting normal and malignant B cells remains incompletely understood. We reported previously that epratuzumab displayed in vitro cytotoxicity to CD22-expressing Burkitt lymphoma cell lines (Daudi and Ramos) only when immobilized on plates or combined with a crosslinking antibody plus a suboptimal amount of anti-IgM (1 µg/mL). Herein, we show that, in the absence of additional anti-IgM ligation, extensive crosslinking of CD22 by plate-immobilized epratuzumab induced intracellular changes in Daudi cells similar to ligating B-cell antigen receptor with a sufficiently high amount of anti-IgM (10 µg/mL). Specifically, either treatment led to phosphorylation of CD22, CD79a and CD79b, along with their translocation to lipid rafts, both of which were essential for effecting caspase-dependent apoptosis. Moreover, such immobilization induced stabilization of F-actin, phosphorylation of Lyn, ERKs and JNKs, generation of reactive oxygen species (ROS), decrease in mitochondria membrane potential (Δψm), upregulation of pro-apoptotic Bax, and downregulation of anti-apoptotic Bcl-xl and Mcl-1. The physiological relevance of immobilized epratuzumab was implicated by noting that several of its in vitro effects, including apoptosis, drop in Δψm, and generation of ROS, could be observed with soluble epratuzumab in Daudi cells co-cultivated with human umbilical vein endothelial cells. These results suggest that the in vivo mechanism of non-ligand-blocking epratuzumab may, in part, involve the unmasking of CD22 to facilitate the trans-interaction of B cells with vascular endothelium.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Apoptose/efeitos dos fármacos , Linfoma de Burkitt/imunologia , Caspases/imunologia , Capeamento Imunológico/efeitos dos fármacos , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos B/antagonistas & inibidores , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Apoptose/imunologia , Linfoma de Burkitt/tratamento farmacológico , Linhagem Celular Tumoral , Humanos , Microdomínios da Membrana/imunologia , Proteínas de Neoplasias/antagonistas & inibidores , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologiaRESUMO
Depletion of B cells with the anti-CD20 antibody rituximab is an established therapy for rheumatoid arthritis. However, rituximab has only moderate efficacy, most likely due to insufficient depletion of B cells in lymphoid organs and expansion of pathogenic B cells. We found that an antibody against mouse CD79b profoundly blocks B-cell proliferation induced via the B-cell receptor, CD40, CD180, and chondroitin sulfate, but not via TLR4 or TLR9. Treatment with anti-CD79b also induces death in resting and activated B cells. B-cell inhibition is mediated by cross-linkage of CD79b, but independent of Fc-receptor engagement. In the model of collagen-induced arthritis, an antibody against mouse CD20 depletes B cells very efficiently but fails to suppress the humoral immune response against collagen and the development of arthritis. In contrast, the antibody against CD79b, and a deglycosylated variant of this antibody, almost completely inhibits the increase in anti-collagen antibodies and the development of arthritis. In mice with established arthritis only the fully glycosylated antibody against CD79b is effective. Our data show that targeting B cells via CD79b is much more effective than B-cell depletion with anti-CD20 antibodies for therapy of arthritis. These findings may have important implications for treatment of B-cell-mediated autoimmune diseases.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Antirreumáticos/farmacologia , Artrite Experimental/tratamento farmacológico , Linfócitos B/imunologia , Antígenos CD79/antagonistas & inibidores , Depleção Linfocítica , Animais , Antígenos CD/imunologia , Artrite Experimental/imunologia , Artrite Experimental/patologia , Linfócitos B/patologia , Antígenos CD40/imunologia , Antígenos CD79/imunologia , Proliferação de Células/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Capeamento Imunológico/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos DBA , Receptores de Antígenos de Linfócitos B/imunologia , RituximabRESUMO
Programmed death-ligand 1 (PD-L1) blockade is accepted as a novel strategy for the reactivation of exhausted T cells that express programmed death-1 (PD-1). However, the mechanism of PD-L1-mediated inhibitory signalling after PD-L1 cross-linking by anti-PD-L1 monoclonal antibody (mAb) or PD-1-immunogloblin fusion protein (PD-1-Ig) is still unknown, although it may induce cell death of PD-L1(+) cells required for regular immune reactions. In this study, PD-1-Ig or anti-PD-L1 mAb treatment was tested in cell lines that expressed PD-L1 and bovine lymphocytes to investigate whether the treatment induces immune reactivation or PD-L1-mediated cell death. PD-L1 cross-linking by PD-1-Ig or anti-PD-L1 mAb primarily increased the number of dead cells in PD-L1(high) cells, but not in PD-L1(low) cells; these cells were prepared from Cos-7 cells in which bovine PD-L1 expression was induced by transfection. The PD-L1-mediated cell death also occurred in Cos-7 and HeLa cells transfected with vectors only encoding the extracellular region of PD-L1. In bovine lymphocytes, the anti-PD-L1 mAb treatment up-regulated interferon-γ (IFN-γ) production, whereas PD-1-Ig treatment decreased this cytokine production and cell proliferation. The IFN-γ production in B-cell-depleted peripheral blood mononuclear cells was not reduced by PD-1-Ig treatment and the percentages of dead cells in PD-L1(+) B cells were increased by PD-1-Ig treatment, indicating that PD-1-Ig-induced immunosuppression in bovine lymphocytes could be caused by PD-L1-mediated B-cell death. This study provides novel information for the understanding of signalling through PD-L1.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Linfócitos B/imunologia , Antígeno B7-H1/imunologia , Tolerância Imunológica/efeitos dos fármacos , Capeamento Imunológico/efeitos dos fármacos , Animais , Antígeno B7-H1/genética , Células CHO , Células COS , Bovinos , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Chlorocebus aethiops , Cricetinae , Cricetulus , Células HeLa , Humanos , Interferon gama/imunologiaRESUMO
Soluble TRAIL (sTRAIL) can be produced by myeloid-derived cells to kill cancer cells. Whether this mechanism is used by T cells, and if so, how sTRAIL production is regulated, remains unclear. Our previous studies showed that ex vivo expanded human γδ T cells express TRAIL and NK receptor group 2 (R2), member D (NKG2D), and possess potent anticancer activities both in vitro and in vivo. Here, we investigated in greater detail the mechanisms by which γδ T cells utilize TRAIL and NKG2D to kill lung cancer cells. We demonstrate that human lung cancer cells express TRAIL R2 and NKG2D ligands. Blocking TRAIL or NKG2D during γδ T-cell-lung cancer cell co-cultures significantly reduced γδ T-cell-mediated cytotoxicity. Cross-linking NKG2D with anti-NKG2D antibody to mimic ligand binding promoted γδ T cells to produce sTRAIL, which induced apoptosis in lung cancer cells through TRAIL R2. Either neutralizing sTRAIL or blocking lung cancer cell TRAIL R2 significantly reduced γδ T-cell-mediated cytotoxicity to lung cancer cells. This study demonstrates that γδ T cells can mediate anticancer immunity via NKG2D-regulated production of sTRAIL.
Assuntos
Imunidade Celular , Neoplasias Pulmonares/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Capeamento Imunológico/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Linfócitos T/patologiaRESUMO
The internalization of matter by phagocytosis is of key importance in the defence against bacterial pathogens and in the control of cancerous tumour growth. Despite the fact that phagocytosis is an inherently mechanical process, little is known about the forces and energies that a cell requires for internalization. Here, we use functionalized magnetic particles as phagocytic targets and track their motion while actuating them in an oscillating magnetic field, in order to measure the translational and rotational stiffnesses of the phagocytic cup as a function of time. The measured evolution of stiffness reveals a characteristic pattern with a pronounced peak preceding the finalization of uptake. The measured stiffness values and their time dependence can be interpreted with a model that describes the phagocytic cup as a prestressed membrane connected to an elastically deformable actin cortex. In the context of this model, the stiffness peak is a direct manifestation of a previously described mechanical bottleneck, and a comparison of model and data suggests that the membrane advances around the particle at a speed of about 20 nm s(-1). This approach is a novel way of measuring the progression of emerging phagocytic cups and their mechanical properties in situ and in real time.
Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Capeamento Imunológico/fisiologia , Nanopartículas de Magnetita , Fagocitose/fisiologia , Linhagem Celular Tumoral , Elasticidade , Humanos , Capeamento Imunológico/efeitos dos fármacos , Fagocitose/efeitos dos fármacosRESUMO
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 , UbiquitinaçãoRESUMO
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/imunologiaRESUMO
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 , SigmodontinaeRESUMO
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/farmacologiaRESUMO
A specialized intercellular junction between podocytes, known as the slit diaphragm (SD), forms the essential structural frame-work for glomerular filtration in the kidney. In addition, mounting evidence demonstrates that the SD also plays a crucial role as a signaling platform in physiological and pathological states. Nephrin, the major component of the SD, is tyrosine-phosphorylated by a Src family tyrosine kinase, Fyn, in developing or injured podocytes, recruiting Nck to Nephrin via its Src homology 2 domain to regulate dynamic actin remodeling. Dysregulated Ca(2+) homeostasis has also been implicated in podocyte damage, but the mechanism of how podocytes respond to injury is largely unknown. Here we have identified phospholipase C-gamma1 (PLC-gamma1) as a novel phospho-Nephrin-binding protein. When HEK293T cells expressing a chimeric protein consisting of CD8 and Nephrin cytoplasmic domain (CD) were treated with anti-CD8 and anti-mouse antibodies, clustering of Nephrin and phosphorylation of Nephrin-CD were induced. Upon this clustering, PLC-gamma1 was bound to phosphorylated Nephrin Tyr-1204, which induced translocation of PLC-gamma1 from cytoplasm to the CD8/Nephrin cluster on the plasma membrane. The recruitment of PLC-gamma1 to Nephrin activated PLC-gamma1, as detected by phosphorylation of PLC-gamma1 Tyr-783 and increase in inositol 1,4,5-trisphosphate level. We also found that Nephrin Tyr-1204 phosphorylation triggers the Ca(2+) response in a PLC-gamma1-dependent fashion. Furthermore, PLC-gamma1 is significantly phosphorylated in injured podocytes in vivo. Given the profound effect of PLC-gamma in diverse cellular functions, regulation of the Ca(2+) signaling by Nephrin may be important in modulating the glomerular filtration barrier function.
Assuntos
Sinalização do Cálcio/fisiologia , Proteínas de Membrana/metabolismo , Fosfolipase C gama/metabolismo , Podócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos/farmacologia , Antígenos CD8/genética , Antígenos CD8/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Capeamento Imunológico/efeitos dos fármacos , Inositol 1,4,5-Trifosfato/genética , Inositol 1,4,5-Trifosfato/metabolismo , Junções Intercelulares/genética , Junções Intercelulares/metabolismo , Proteínas de Membrana/genética , Camundongos , Células NIH 3T3 , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fosfolipase C gama/genética , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Podócitos/citologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Posttranscriptional regulation of gene expression plays a pivotal role as a fast control system for T-cells and B-cells operating in the defense reactions against rapidly growing infectious agents. The framework of this machinery involves cis-acting elements in the mRNAs of relevant cytokines and trans-acting factors interacting with these elements. The cis- and trans-acting factors enforce rapid mRNA decay with other proteins such as nucleases in the decay machinery. The most prominent cis-element contains A + U- rich sequence (ARE), and is located in the 3'-untranslated region of the target mRNAs. Some ARE-binding proteins promote the rapid decay, and others protect the mRNA from degradation. The 5'-end of nascent mRNA undergoes capping which protects the 5'-end together with the cap-binding protein, and the 3' end is protected with poly (A) tail and associating poly (A) binding protein. Unlike in classical drawing of linear structure of mRNA, the end structures interact with each other through a common platform composed of translation initiation factors, revealing the cross-talk of the 5'-end cap structure and 3'-end poly (A) tail on the translational machinery. The rapid degradation and stabilization of mRNA is triggered by a cellular signaling cascade through phosphorylation of associating protein factors in response to environmental stimuli, and a large nucleolytic complex for specific decay reaction called exosome is formed with the 3'-UTR of mRNA through interaction with the ARE-binding proteins. Possible therapeutic agents modifying stability of ARE-containing mRNA are being screened in order to treat immunological disorders.
Assuntos
Anti-Infecciosos/farmacologia , Anti-Infecciosos/uso terapêutico , Infecções/tratamento farmacológico , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/fisiologia , Adenina/metabolismo , Animais , Citocinas/biossíntese , Citocinas/genética , Humanos , Capeamento Imunológico/efeitos dos fármacos , Ligação Proteica , RNA Mensageiro/biossíntese , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
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/imunologiaRESUMO
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 U937RESUMO
CD47 functions as a marker of self on red blood cells (RBCs) by binding to signal regulatory protein alpha on macrophages, preventing phagocytosis of autologous RBCs by splenic red pulp macrophages, and Fcgamma receptor (FcgammaR)- or complement receptor-mediated phagocytosis by macrophages in general. RBC senescence involves a series of biochemical changes to plasma membrane proteins or lipids, which may regulate phagocytosis by macrophages. Here, we investigated whether CD47 on experimentally senescent murine RBCs affects their phagocytosis by macrophages in vitro. Clustering of CD47 with antibodies was more pronounced in the plasma membrane of untreated RBCs, compared with that in in vitro oxidized RBCs (Ox-RBCs). Phagocytosis of Ox-RBCs was mediated by scavenger receptors (SRs) distinct from SR-A or CD36 and required serum factors. We found that wild-type (WT) and CD47(-/-) Ox-RBCs were phagocytosed equally well by macrophages in the presence of serum, suggesting that phagocytosis via SRs is not inhibited by CD47. Despite this, FcgammaR-mediated phagocytosis of IgG-opsonized Ox-RBCs was strongly inhibited by CD47. These data suggest that based on the specific prophagocytic receptors mediating uptake of senescent RBCs, the phagocytosis-inhibitory role of CD47 may be more or less involved.
Assuntos
Antígeno CD47/metabolismo , Senescência Celular/fisiologia , Membrana Eritrocítica/metabolismo , Macrófagos/metabolismo , Fagocitose/fisiologia , Receptores de IgG/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antígeno CD47/genética , Senescência Celular/efeitos dos fármacos , Membrana Eritrocítica/genética , Capeamento Imunológico/efeitos dos fármacos , Capeamento Imunológico/fisiologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Oxirredução , Fagocitose/efeitos dos fármacos , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Receptores de IgG/genética , Receptores Imunológicos , Receptores Depuradores , Baço/citologia , Baço/metabolismoRESUMO
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 LinfócitosRESUMO
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/imunologiaRESUMO
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 SrcRESUMO
Platelet alpha-granules constitute the major rapidly releasable reservoir of thrombospondin-1 in higher animals. Although some fragments and peptides derived from thrombospondin-1 stimulate or inhibit platelet aggregation, its physiologic function in platelets has remained elusive. We now show that endogenous thrombospondin-1 is necessary for platelet aggregation in vitro in the presence of physiologic levels of nitric oxide (NO). Exogenous NO or elevation of cGMP delays thrombin-induced platelet aggregation under high shear and static conditions, and exogenous thrombospondin-1 reverses this delay. Thrombospondin-1-null murine platelets fail to aggregate in response to thrombin in the presence of exogenous NO or 8Br-cGMP. At physiologic concentrations of the NO synthase substrate arginine, thrombospondin-1-null platelets have elevated basal cGMP. Ligation of CD36 or CD47 is sufficient to block NO-induced cGMP accumulation and mimic the effect of thrombospondin-1 on aggregation. Exogenous thrombospondin-1 also reverses the suppression by NO of alphaIIb/beta3 integrin-mediated platelet adhesion on immobilized fibrinogen, mediated in part by increased GTP loading of Rap1. Thrombospondin-1 also inhibits cGMP-mediated activation of cGMP-dependent protein kinase and thereby prevents phosphorylation of VASP. Thus, release of thrombospondin-1 from alpha-granules during activation provides positive feedback to promote efficient platelet aggregation and adhesion by overcoming the antithrombotic activity of physiologic NO.
Assuntos
Plaquetas/metabolismo , GMP Cíclico/metabolismo , Fibrinolíticos/metabolismo , Óxido Nítrico/metabolismo , Agregação Plaquetária/fisiologia , Trombospondina 1/metabolismo , Animais , Arginina/genética , Arginina/metabolismo , Plaquetas/citologia , Antígenos CD36/genética , Antígenos CD36/metabolismo , Antígeno CD47/genética , Antígeno CD47/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , GMP Cíclico/antagonistas & inibidores , GMP Cíclico/genética , GMP Cíclico/farmacologia , Fibrinolíticos/farmacologia , Capeamento Imunológico/efeitos dos fármacos , Capeamento Imunológico/fisiologia , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/genética , Peptídeos/genética , Peptídeos/metabolismo , Peptídeos/farmacologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Adesividade Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/genética , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Vesículas Secretórias/genética , Vesículas Secretórias/metabolismo , Resistência ao Cisalhamento , Trombina/genética , Trombina/metabolismo , Trombina/farmacologia , Trombospondina 1/genética , Trombospondina 1/farmacologia , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
We investigated the enzymes responsible for FcepsilonRI-dependent production of reactive oxygen species (ROS) and the influence of ROS on mast cell secretory responses. 5-Lipoxygenase (5-LO) was the primary enzyme involved in ROS production by human mast cells (huMC) and mouse bone marrow-derived mast cells (mBMMC) following FcepsilonRI aggregation because incubation with 5-LO inhibitors (AA861, nordihydroguaiaretic acid, zileuton) but not a flavoenzyme inhibitor (diphenyleneiodonium) completely abrogated Ag-induced dichlorodihydrofluorescein (DCF) fluorescence. Furthermore, 5-LO-deficient mBMMC had greatly reduced FcepsilonRI-dependent DCF fluorescence compared with wild type mBMMC or those lacking a functional NADPH oxidase (i.e., gp91(phox)- or p47(phox)-deficient cells). A minor role for cyclooxygenase (COX)-1 in FcepsilonRI-dependent ROS production was demonstrated by inhibition of Ag-mediated DCF fluorescence by a COX-1 inhibitor (FR122047) and reduced DCF fluorescence in COX-1-deficient mBMMC. Complete abrogation of FcepsilonRI-dependent ROS production in mast cells had no effect on degranulation or cytokine secretion. In response to the NADPH oxidase-stimulating agents including PMA, mBMMC and huMC produced negligible ROS. IgG-coated latex beads did stimulate ROS production in huMC, and in this experiment 5-LO and COX again appeared to be the enzymatic sources of ROS. In contrast, IgG-coated latex bead-induced ROS production in human polymorphonuclear leukocytes occurred by the NADPH oxidase pathway. Thus mBMMC and huMC generate ROS by 5-LO and COX-1 in response to FcepsilonRI aggregation; huMC generate ROS upon exposure to IgG-coated latex beads by 5-LO and COX; and ROS appear to have no significant role in FcepsilonRI-dependent degranulation and cytokine production.