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1.
Eur J Immunol ; 33(2): 326-33, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12548563

ABSTRACT

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.


Subject(s)
Bacterial Outer Membrane Proteins/pharmacology , Chemokines, CC/pharmacology , Dendritic Cells/drug effects , Gene Expression Regulation/drug effects , Lymphoid Tissue/cytology , Macrophages/drug effects , Adoptive Transfer , Animals , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Cell Movement/drug effects , Chemokine CCL19 , Chemokine CCL21 , Chemokine CCL3 , Chemokine CCL4 , Chemokine CCL5/biosynthesis , Chemokine CCL5/genetics , Chemokines, CC/biosynthesis , Chemokines, CC/genetics , Dendritic Cells/cytology , Dendritic Cells/immunology , Down-Regulation/drug effects , Humans , Inflammation/chemically induced , Interleukin-8/biosynthesis , Interleukin-8/genetics , Lipopolysaccharides/pharmacology , Lymph Nodes/pathology , Macrophage Inflammatory Proteins/biosynthesis , Macrophage Inflammatory Proteins/genetics , Macrophages/cytology , Macrophages/immunology , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Receptors, CCR1 , Receptors, CCR5/biosynthesis , Receptors, CCR5/genetics , Receptors, CCR7 , Receptors, Chemokine/biosynthesis , Receptors, Chemokine/genetics , Up-Regulation/drug effects
2.
Blood ; 101(1): 143-50, 2003 Jan 01.
Article in English | MEDLINE | ID: mdl-12393446

ABSTRACT

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.


Subject(s)
Autocrine Communication , Dendritic Cells/cytology , Interferon-gamma/physiology , Macrophages/cytology , Monocytes/cytology , Animals , Antigens, CD/analysis , Bone Marrow Cells/cytology , Cell Culture Techniques/methods , Cell Differentiation/drug effects , Cytokines/pharmacology , Gene Expression Regulation/drug effects , Hematopoietic Stem Cells/drug effects , Immunophenotyping , Interferon-gamma/pharmacology , Interleukin-6/biosynthesis , Interleukin-6/pharmacology , Macrophage Colony-Stimulating Factor/biosynthesis , Macrophage Colony-Stimulating Factor/drug effects , Macrophage Colony-Stimulating Factor/pharmacology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL
3.
Cytometry ; 48(2): 71-9, 2002 Jun 01.
Article in English | MEDLINE | ID: mdl-12116367

ABSTRACT

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.


Subject(s)
Dendritic Cells/metabolism , Flow Cytometry/methods , Image Cytometry/methods , Microscopy, Confocal , NF-kappa B/metabolism , Animals , Cell Nucleus/drug effects , Cell Nucleus/immunology , Cell Nucleus/metabolism , Cell Separation , Cells, Cultured , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dose-Response Relationship, Immunologic , Escherichia coli/immunology , Humans , Lipopolysaccharides/pharmacology , Lymphocyte Activation , Mice , Reproducibility of Results
4.
J Immunol ; 168(12): 6366-74, 2002 Jun 15.
Article in English | MEDLINE | ID: mdl-12055254

ABSTRACT

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.


Subject(s)
Chagas Disease/mortality , Chagas Disease/prevention & control , Dendritic Cells/cytology , Drosophila Proteins , Membrane Glycoproteins/physiology , Protein Disulfide Reductase (Glutathione)/physiology , Protozoan Proteins/physiology , Receptors, Cell Surface/physiology , Signal Transduction/immunology , Trypanosoma cruzi/enzymology , Animals , Binding Sites/immunology , Cell Differentiation/immunology , Cells, Cultured , Chagas Disease/enzymology , Chagas Disease/immunology , Dendritic Cells/metabolism , Glutathione/metabolism , Humans , Injections, Intraperitoneal , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Protein Binding/immunology , Protein Disulfide Reductase (Glutathione)/administration & dosage , Protein Disulfide Reductase (Glutathione)/immunology , Protein Disulfide Reductase (Glutathione)/metabolism , Protozoan Proteins/administration & dosage , Protozoan Proteins/immunology , Protozoan Proteins/metabolism , Protozoan Vaccines/administration & dosage , Protozoan Vaccines/immunology , Toll-Like Receptor 2 , Toll-Like Receptors , Trypanosoma cruzi/immunology
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