Differential roles of PI3-Kinase, MAPKs and NF-kappaB on the manipulation of dendritic cell T(h)1/T(h)2 cytokine/chemokine polarizing profile.
Dendritic cells (DC) are professional antigen-presenting cells with a unique capacity to initiate and modulate immune responses by their ability to prime naïve T-cells. Upon stimuli, DC experience several morphologic, phenotypic and functional changes in a process referred to as maturation. This process is crucial to the biological functions of DC since their maturation status confer them the ability to polarize distinct T-cell subsets. In this work we explored the relevance of PI3-Kinase, Mitogen-Activated Protein Kinases (MAPKs) and NF-kappaB on cytokines/chemokines and co-stimulatory molecules expression. As experimental model, we used a fetal skin-derived dendritic cell line (FSDC) induced to mature by treatment with lipopolysaccharide (LPS). Morphology and ultrastructure were analyzed by confocal and electron microscopies, respectively. Levels of phosphorylated proteins were evaluated by Western blot, production of cytokines/chemokines was analyzed by protein arrays and the expression of surface molecules was evaluated by flow cytometry. The effect of specific inhibitors of the studied signaling pathways on the transcription of cytokines/chemokines and co-stimulatory molecules was accessed by Quantitative Real-Time RT-PCR. The results showed that LPS induces significant morphological and ultrastructural changes in FSDC. Western blot analysis revealed that LPS challenge promotes an early and transient activation of NF-kappaB, ERK1/2, p38 MAPK, along with a more sustained PI3 kinase/AKT activation. The co-stimulatory CD40, CD80, CD86 and antigen-presenting MHC class I and II molecules were increased and among secreted molecules, interleukin IL-6, CCL5, G-CSF, CCL2, CXCL2 were strongly up-regulated. Using a pharmacological approach we observed that LPS-induced increase of these molecules was differentially regulated by the distinct signaling pathways. Moreover, the polarizing T(h)2 cytokines/chemokines induced by LPS in FSDC were found to be positively regulated by NF-kappaB and ERK and negatively modulated by p38 MAPK. Altogether these results suggest that the use of pharmacological inhibitors to manipulate DC maturation, namely the polarizing T(h)1/T(h)2 cytokine/chemokine profile, may be useful in the development of more specific immunotherapeutic protocols.
Neves
Bruno Miguel
BM
Universidade de Coimbra, Portugal.
Cruz
Maria Teresa
MT
Francisco
Vera
V
Garcia-Rodriguez
Cármen
C
Silvestre
Ricardo
R
Cordeiro-da-Silva
Anabela
A
Dinis
Augusto M
AM
Batista
Maria Teresa
MT
Duarte
Carlos B
CB
Lopes
Maria Celeste
MC
D000818:Animals; D018122:B7-1 Antigen / Q000235:genetics / Q000378:metabolism; D051940:B7-2 Antigen / Q000235:genetics / Q000378:metabolism; D026481:Blotting, Far-Western; D002460:Cell Line; D018925:Chemokines / Q000235:genetics / Q000378:metabolism*; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D003713:Dendritic Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006649:Histocompatibility Antigens / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018417:Th1 Cells / Q000378:metabolism; D018418:Th2 Cells / Q000378:metabolism
D018122:B7-1 Antigen; D051940:B7-2 Antigen; D018925:Chemokines; D016207:Cytokines; D006649:Histocompatibility Antigens; D008070:Lipopolysaccharides; D016328:NF-kappa B; D019869:Phosphatidylinositol 3-Kinases; D020928:Mitogen-Activated Protein Kinases