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1.
Oncoimmunology ; 5(7): e1178420, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27622031

RESUMO

Despite the recent approval of new agents for metastatic melanoma, its treatment remains challenging. Moreover, few available immunotherapies induce a strong cellular immune response, and selection of the correct immunoadjuvant is crucial for overcoming this obstacle. Here, we studied the immunomodulatory properties of arazyme, a bacterial metalloprotease, which was previously shown to control metastasis in a murine melanoma B16F10-Nex2 model. The antitumor activity of arazyme was independent of its proteolytic activity, since heat-inactivated protease showed comparable properties to the active enzyme; however, the effect was dependent on an intact immune system, as antitumor properties were lost in immunodeficient mice. The protective response was IFNγ-dependent, and CD8(+) T lymphocytes were the main effector antitumor population, although B and CD4(+) T lymphocytes were also induced. Macrophages and dendritic cells were involved in the induction of the antitumor response, as arazyme activation of these cells increased both the expression of surface activation markers and proinflammatory cytokine secretion through TLR4-MyD88-TRIF-dependent, but also MAPK-dependent pathways. Arazyme was also effective in the murine breast adenocarcinoma 4T1 model, reducing primary and metastatic tumor development, and prolonging survival. To our knowledge, this is the first report of a bacterial metalloprotease interaction with TLR4 and subsequent receptor activation that promotes a proinflammatory and tumor protective response. Our results show that arazyme has immunomodulatory properties, and could be a promising novel alternative for metastatic melanoma treatment.

2.
Blood ; 119(7): 1747-56, 2012 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-22186994

RESUMO

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation, and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the hematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other hematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that depends on CLEC-2 and Syk. These studies found that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behavior in vitro.


Assuntos
Plaquetas/metabolismo , Linhagem da Célula/genética , Crescimento e Desenvolvimento/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Lectinas Tipo C/fisiologia , Megacariócitos/metabolismo , Proteínas Tirosina Quinases/fisiologia , Animais , Animais Recém-Nascidos , Plaquetas/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Células Cultivadas , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Crescimento e Desenvolvimento/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Megacariócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Quinase Syk , Trombopoese/genética , Trombopoese/fisiologia
3.
Eur J Immunol ; 41(10): 3040-53, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21728173

RESUMO

Myeloid cells express a plethora of C-type lectin receptors (CLRs) that can regulate immune responses. CLEC-2 belongs to the Dectin-1 sub-family of CLRs that possess an extracellular C-type lectin-like domain and a single intracellular hemITAM motif. CLEC-2 is highly expressed on mouse and human platelets where it signals via Syk to promote aggregation. We generated a monoclonal antibody (mAb) against mouse CLEC-2 and found that CLEC-2 is additionally widely expressed on leukocytes and that its expression is upregulated during inflammation. MAb-mediated crosslinking of CLEC-2 leads to hemITAM-dependent signaling via Syk, Ca(2+) and NFAT and, in myeloid cells, modulates the effect of toll-like receptor (TLR) agonists to selectively potentiate production of IL-10. A macrophage/dendritic cell-dependent increase in IL-10 is also observed in mice given anti-CLEC-2 mAb together with LPS. Collectively, these data indicate that CLEC-2 is expressed in myeloid cells and acts as a Syk-coupled CLR able to modulate TLR signaling and inflammatory responses.


Assuntos
Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/metabolismo , Células Mieloides/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Anticorpos Monoclonais , Plaquetas , Cálcio/metabolismo , Linhagem Celular , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Dinoprostona/metabolismo , Inflamação , Mediadores da Inflamação/imunologia , Interleucina-10/biossíntese , Lectinas Tipo C/biossíntese , Lectinas Tipo C/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/imunologia , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais , Quinase Syk , Receptores Toll-Like/agonistas
4.
J Biol Chem ; 286(6): 4107-16, 2011 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-21098033

RESUMO

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Motivos de Aminoácidos , Animais , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lectinas Tipo C/genética , Camundongos , Camundongos Knockout , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas Tirosina Quinases/genética , Pirimidinas/farmacologia , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinase Syk , Venenos de Víboras/farmacologia
5.
J Immunol ; 182(7): 4150-7, 2009 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19299712

RESUMO

CLEC-2 is a member of the "dectin-1 cluster" of C-type lectin-like receptors and was originally thought to be restricted to platelets. In this study, we demonstrate that murine CLEC-2 is also expressed by peripheral blood neutrophils, but only weakly by bone marrow or elicited inflammatory neutrophils. On circulating neutrophils, CLEC-2 can mediate phagocytosis of Ab-coated beads and the production of proinflammatory cytokines, including TNF-alpha, in response to the CLEC-2 ligand, rhodocytin. CLEC-2 possesses a tyrosine-based cytoplasmic motif similar to that of dectin-1, and we show using chimeric analyses that the activities of this receptor are dependent on this tyrosine. Like dectin-1, CLEC-2 can recruit the signaling kinase Syk in myeloid cells, however, stimulation of this pathway does not induce the respiratory burst. These data therefore demonstrate that CLEC-2 expression is not restricted to platelets and that it functions as an activation receptor on neutrophils.


Assuntos
Lectinas Tipo C/biossíntese , Neutrófilos/metabolismo , Fagocitose/fisiologia , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Expressão Gênica , Regulação da Expressão Gênica , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/genética , Camundongos , Neutrófilos/imunologia , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Explosão Respiratória/imunologia , Quinase Syk , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologia
6.
FASEB J ; 23(4): 1262-71, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19088181

RESUMO

Macrophage migration inhibitory factor (MIF) participates in the pathogenesis of inflammatory diseases, including asthma, in which it enhances airway hypersensitivity and tissue eosinophilia. Herein, we investigated the role of MIF in eosinophilopoiesis and tissue eosinophilia using Schistosoma mansoni infection. MIF-deficient (Mif(-/-)) mice had similar numbers of adult worms, eggs, and granulomas compared to wild-type mice, but the size of granulomas was strikingly reduced due to smaller numbers of eosinophils. MIF did not affect the acquired response to infection, as Mif(-/-) mice produced normal amounts of Th2 cytokines and IgE. Nevertheless, recombinant MIF (rMIF) behaved as a chemoattractant for eosinophils, what could partially explain the reduced eosinophilia in infected Mif(-/-) mice. Moreover, the percentage of eosinophils was reduced in bone marrows of Mif(-/-) mice chronically infected with S. mansoni compared to wild type. Mif(-/-) had impaired eosinophilopoiesis in response to interleukin (IL)-5 and addition of rMIF to bone marrow cultures from IL-5 transgenic mice enhanced the generation of eosinophils. In the absence of MIF, eosinophil precursors were unable to survive the IL-5-supplemented cell culture, and were ingested by macrophages. Treatment with pancaspase inhibitor z-VAD or rMIF promoted the survival of eosinophil progenitors. Together, these results indicate that MIF participates in IL-5-driven maturation of eosinophils and in tissue eosinophilia associated with S. mansoni infection.


Assuntos
Eosinofilia/imunologia , Eosinófilos/imunologia , Interleucina-5/fisiologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Esquistossomose mansoni/patologia , Animais , Eosinofilia/etiologia , Eosinofilia/patologia , Eosinófilos/patologia , Granuloma/etiologia , Granuloma/imunologia , Granuloma/patologia , Inflamação/patologia , Interleucina-5/imunologia , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Recombinantes/imunologia , Esquistossomose mansoni/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/patologia
7.
J Clin Invest ; 118(6): 2098-110, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18497879

RESUMO

The mouse CD8alpha+ DC subset excels at cross-presentation of antigen, which can elicit robust CTL responses. A receptor allowing specific antigen targeting to this subset and its equivalent in humans would therefore be useful for the induction of antitumor CTLs. Here, we have characterized a C-type lectin of the NK cell receptor group that we named DC, NK lectin group receptor-1 (DNGR-1). DNGR-1 was found to be expressed in mice at high levels by CD8+ DCs and at low levels by plasmacytoid DCs but not by other hematopoietic cells. Human DNGR-1 was also restricted in expression to a small subset of blood DCs that bear similarities to mouse CD8alpha+ DCs. The selective expression pattern and observed endocytic activity of DNGR-1 suggested that it could be used for antigen targeting to DCs. Consistent with this notion, antigen epitopes covalently coupled to an antibody specific for mouse DNGR-1 were selectively cross-presented by CD8alpha+ DCs in vivo and, when given with adjuvants, induced potent CTL responses. When the antigens corresponded to tumor-expressed peptides, treatment with the antibody conjugate and adjuvant could prevent development or mediate eradication of B16 melanoma lung pseudometastases. We conclude that DNGR-1 is a novel, highly specific marker of mouse and human DC subsets that can be exploited for CTL cross-priming and tumor therapy.


Assuntos
Antígenos CD8/biossíntese , Células Dendríticas/citologia , Imunoterapia/métodos , Lectinas/metabolismo , Melanoma/patologia , Melanoma/terapia , Neoplasias/terapia , Receptores Imunológicos/metabolismo , Linfócitos T Citotóxicos/metabolismo , Animais , Antígenos/química , Células Dendríticas/imunologia , Humanos , Lectinas Tipo C , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Camundongos , Modelos Biológicos , Metástase Neoplásica , Receptores Imunológicos/genética , Receptores Imunológicos/fisiologia , Receptores Mitogênicos
8.
J Biol Chem ; 282(28): 20221-9, 2007 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-17502383

RESUMO

Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an atom of iron linked to four ligand groups of porphyrin. A high amount of free heme, a potential amplifier of the inflammatory response, is a characteristic feature of diseases with increased hemolysis or extensive cell damage. Here we demonstrate that heme, but not its analogs/precursors, induced tumor necrosis factor-alpha (TNF-alpha) secretion by macrophages dependently on MyD88, TLR4, and CD14. The activation of TLR4 by heme is exquisitely strict, requiring its coordinated iron and the vinyl groups of the porphyrin ring. Signaling of heme through TLR4 depended on an interaction distinct from the one established between TLR4 and lipopolysaccharide (LPS) since anti-TLR4/MD2 antibody or a lipid A antagonist inhibited LPS-induced TNF-alpha secretion but not heme activity. Conversely, protoporphyrin IX antagonized heme without affecting LPS-induced activation. Moreover, heme induced TNF-alpha and keratinocyte chemokine but was ineffective to induce interleukin-6, interleukin-12, and interferon-inducible protein-10 secretion or co-stimulatory molecule expression. These findings support the concept that the broad ligand specificity of TLR4 and the different activation profiles might in part reside in its ability to recognize different ligands in different binding sites. Finally, heme induced oxidative burst, neutrophil recruitment, and heme oxygenase-1 expression independently of TLR4. Thus, our results presented here reveal a previous unrecognized role of heme as an extracellular signaling molecule that affects the innate immune response through a receptor-mediated mechanism.


Assuntos
Heme/imunologia , Imunidade Inata/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Células Cultivadas , Citocinas/imunologia , Antagonismo de Drogas , Regulação Enzimológica da Expressão Gênica , Heme/antagonistas & inibidores , Heme/farmacologia , Heme Oxigenase-1/imunologia , Hemólise/genética , Hemólise/imunologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Inflamação/genética , Inflamação/imunologia , Receptores de Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/genética , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/imunologia , Neutrófilos/imunologia , Fármacos Fotossensibilizantes/antagonistas & inibidores , Fármacos Fotossensibilizantes/farmacologia , Protoporfirinas/antagonistas & inibidores , Protoporfirinas/farmacologia , Explosão Respiratória/efeitos dos fármacos , Explosão Respiratória/genética , Explosão Respiratória/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Receptor 4 Toll-Like/deficiência
9.
Eur J Immunol ; 37(4): 1097-106, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17373669

RESUMO

Macrophage migration inhibitory factor (MIF) is increased in asthmatic patients and plays a critical role in the pathogenesis of asthma. We show here that mice lacking MIF failed to develop airway hyper-responsiveness (AHR), tissue eosinophilia, and mucus metaplasia. Analysis of the bronchoalveolar fluids revealed a substantial reduction of IL-13, eotaxin and cysteinyl-leukotrienes. The lack of these cardinal features of asthma in MIF(-/-) mice occurs regardless of high concentrations of IL-4 in the lung and OVA-specific IgE in the serum. Antigen-specific lymphocyte proliferation and IL-13 production were similarly increased in the draining lymph nodes of OVA-immunized and challenged MIF(-/-) mice compared to WT, but were reduced in the spleen of MIF(-/-), thus indicating differential roles of MIF in these compartments. Stimulation of naive CD4(+) cells with anti-CD3 antibody demonstrated that MIF(-/-) cells produced increased amounts of IFN-gamma and IL-4 compared to WT CD4(+) cells. Finally, treatment of sensitized BALB/c mice with neutralizing anti-MIF antibody abrogated the development of ARH and airway inflammation without affecting the production of Th2 cytokines or IgE. The present study demonstrates that MIF is required for allergic inflammation, adding important elements to our knowledge of asthma pathogenesis and suggesting that neutralization of MIF might be of therapeutic value in asthma.


Assuntos
Alérgenos/imunologia , Asma/metabolismo , Diferenciação Celular/imunologia , Oxirredutases Intramoleculares/fisiologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Células Th2/citologia , Animais , Asma/imunologia , Soros Imunes/administração & dosagem , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/imunologia , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Muco/metabolismo , Células Th2/imunologia , Células Th2/metabolismo
10.
J Biol Chem ; 281(32): 22614-23, 2006 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-16766532

RESUMO

The host response to fungi is in part dependent on activation of evolutionarily conserved receptors, including toll-like receptors and phagocytic receptors. However, the molecular nature of fungal ligands responsible for this activation is largely unknown. Herein, we describe the isolation and structural characterization of an alpha-glucan from Pseudallescheria boydii cell wall and evaluate its role in the induction of innate immune response. These analyses indicate that alpha-glucan of P. boydii is a glycogen-like polysaccharide consisting of linear 4-linked alpha-D-Glcp residues substituted at position 6 with alpha-D-Glcp branches. Soluble alpha-glucan, but not beta-glucan, led to a dose-dependent inhibition of conidia phagocytosis. Furthermore, a significant decrease in the phagocytic index occurred when alpha-glucan from conidial surface was removed by enzymatic treatment with alpha-amyloglucosidase, thus indicating an essential role of alpha-glucan in P. boydii internalization by macrophages. alpha-Glucan stimulates the secretion of inflammatory cytokines by macrophages and dendritic cells; again this effect is abolished by treatment with alpha-amyloglucosidase. Finally, alpha-glucan induces cytokine secretion by cells of the innate immune system in a mechanism involving toll-like receptor 2, CD14, and MyD88. These results might have relevance in the context of infections with P. boydii and other fungi, and alpha-glucan could be a target for intervention during fungal infections.


Assuntos
Glucanos/química , Pseudallescheria/metabolismo , Receptores Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Sequência de Carboidratos , Células Dendríticas/citologia , Receptores de Lipopolissacarídeos/biossíntese , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fator 88 de Diferenciação Mieloide , Fagocitose
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