RESUMO
Outer membrane protein A (OmpA) is a class of bacterial cell wall protein that is immunogenic without adjuvant. As specific immune responses are initiated in the lymph nodes (LN, we analyzed the effect of the OmpA from Klebsiella pneumoniae (KpOmpA) onchemokine/ chemokine receptor expression by APC and on cell migration to the LN. Upon contact with KpOmpA, human immature DC and macrophages acquire CCR7 expression and responsiveness to CCL21. In parallel, CCR1 and CCR5 expression is down-regulated and CXCL8, CCL2, CCL3 and CCL5 production is up-regulated. Mice injected subcutaneously with KpOmpA present a transient inflammatory reaction at the site of injection accompanied by an enlargement of the draining LN with a higher proportion of DC and macrophages. Lastly, when exposed to KpOmpA prior injection, DC but not macrophages migrate to the draining LN. In conclusion, KpOmpA confers a migratory phenotype to DC and triggers their migration to the regional LN. This property contributes to explain how innate cells initiate adaptive immune response upon recognition of conserved bacterial components and also why OmpA is immunogenic in the absence of adjuvant.
Assuntos
Proteínas da Membrana Bacteriana Externa/farmacologia , Quimiocinas CC/farmacologia , Células Dendríticas/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Tecido Linfoide/citologia , Macrófagos/efeitos dos fármacos , Transferência Adotiva , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Movimento Celular/efeitos dos fármacos , Quimiocina CCL19 , Quimiocina CCL21 , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL5/biossíntese , Quimiocina CCL5/genética , Quimiocinas CC/biossíntese , Quimiocinas CC/genética , Células Dendríticas/citologia , Células Dendríticas/imunologia , Regulação para Baixo/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Interleucina-8/biossíntese , Interleucina-8/genética , Lipopolissacarídeos/farmacologia , Linfonodos/patologia , Proteínas Inflamatórias de Macrófagos/biossíntese , Proteínas Inflamatórias de Macrófagos/genética , Macrófagos/citologia , Macrófagos/imunologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptores CCR1 , Receptores CCR5/biossíntese , Receptores CCR5/genética , Receptores CCR7 , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Human monocytes differentiate into dendritic cells (DCs) or macrophages according to the nature of environmental signals. Monocytes stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin 4 (IL-4) yield DCs. We tested here whether interferon-gamma (IFN-gamma), a potent activator of macrophages, may modulate monocyte differentiation. Addition of IFN-gamma to IL-4 plus GM-CSF-stimulated monocytes switches their differentiation from DCs to CD14(-)CD64(+) macrophages. IFN-gamma increases macrophage colony-stimulating factor (M-CSF) and IL-6 production by IL-4 plus GM-CSF-stimulated monocytes by acting at the transcriptional level and acts together with IL-4 to up-regulate M-CSF but not IL-6 production. IFN-gamma also increases M-CSF receptor internalization. Results from neutralizing experiments show that both M-CSF and IL-6 are involved in the ability of IFN-gamma to skew monocyte differentiation from DCs to macrophages. Finally, this effect of IFN-gamma is limited to early stages of differentiation. When added to immature DCs, IFN-gamma up-regulates IL-6 but not M-CSF production and does not convert them to macrophages, even in the presence of exogenous M-CSF. In conclusion, IFN-gamma shifts monocyte differentiation to macrophages rather than DCs through autocrine M-CSF and IL-6 production. These data show that IFN-gamma controls the differentiation of antigen-presenting cells and thereby reveals a new mechanism by which IFN-gamma orchestrates the outcome of specific immune responses.
Assuntos
Comunicação Autócrina , Células Dendríticas/citologia , Interferon gama/fisiologia , Macrófagos/citologia , Monócitos/citologia , Animais , Antígenos CD/análise , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Citocinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Imunofenotipagem , Interferon gama/farmacologia , Interleucina-6/biossíntese , Interleucina-6/farmacologia , Fator Estimulador de Colônias de Macrófagos/biossíntese , Fator Estimulador de Colônias de Macrófagos/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Nuclear factor kappa B (NF-kappaB) is a ubiquitously expressed transcription factor that regulates cytokine and immunoglobulin (Ig) gene expression. In most cell types, the inactive p50/p65 NF-kappaB heterodimer is located in the cytoplasm, complexed to its IkappaB inhibitory unit. Stimulation of cells by various reagents such as bacterial endotoxin or cytokines leads to a dissociation of NF-kappaB from IkappaB and a rapid translocation of free NF-kappaB to the nucleus. The aim of this article is to define optimal conditions for the measurement of NF-kappaB translocation by both confocal microscopy and flow cytometry. METHODS: Four commercial anti-NF-kappaB antibodies were evaluated by confocal microscopy, after using two methods of fixation and permeabilization of the cells. These antibodies were examined further by flow cytometry on purified nuclei. RESULTS: Paraformaldehyde-methanol treatment of dendritic cells is a good combination to visualize NF-kappaB translocation by confocal microscopy. Three of the four antibodies tested gave good results on nonactivated and on lipopolysaccharide (LPS)-activated dendritic cells. The measurement of NF-kappaB translocation by flow cytometry on purified nuclei is a quick and sensitive method. Only one of the four evaluated antibodies showed a significant difference between nonactivated and activated cells. CONCLUSIONS; Microscopy and flow cytometry are quick and reproducible methods to measure NF-kappaB translocation and can be adapted to identify new molecules that activate dendritic cells.
Assuntos
Células Dendríticas/metabolismo , Citometria de Fluxo/métodos , Citometria por Imagem/métodos , Microscopia Confocal , NF-kappa B/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Separação Celular , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Relação Dose-Resposta Imunológica , Escherichia coli/imunologia , Humanos , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Camundongos , Reprodutibilidade dos TestesRESUMO
The intracellular protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas disease. We have recently identified a T. cruzi-released protein related to thiol-disulfide oxidoreductase family, called Tc52, which is crucial for parasite survival and virulence. In vitro, Tc52 in combination with IFN-gamma activates human macrophages. In vivo, active immunization with Tc52 relieves the immunosuppression associated to acute infection and elicits a specific immune response. As dendritic cells (DC) have a central role in the initiation of immune responses, we investigated whether Tc52 may modulate DC activity. We show that Tc52 induces human DC maturation. Tc52-treated immature DC acquire CD83 and CD86 expression, produce inflammatory chemokines (IL-8, monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1 alpha), and present potent costimulatory properties. Tc52 binds to DC by a mechanism with the characteristics of a saturable receptor system and signals via Toll-like receptor 2. While Tc52-mediated signaling involves its reduced glutathione-binding site, another portion of the molecule is involved in Tc52 binding to DC. Finally, we report that immunization with Tc52 protects mice in vivo against lethal infection with T. cruzi. Together these data evidence complex molecular interactions between the T. cruzi-derived molecule, Tc52, and DC, and suggest that Tc52 and related class of proteins might represent a new type of pathogen-associated molecular patterns. Moreover, the immune protection data suggest that Tc52 is among candidate molecules that may be used to design an optimal multicomponent vaccine to control T. cruzi infection.