Flow cytometry used for the analysis of calcium signaling induced by antigen-specific T-cell activation

Background: In this study, the effect of antigen-presentingcells (APC), peptide concentration, and CD28 costimulationon calcium signaling, induced by antigen-specificT-cell activation, was studied by flow cytometry.Methods: We used two experimental approaches, whichdiffered in their time scale and in the duration of the Tcell-APC interaction, to measure the increase of intracellularfree calcium levels ([Ca2]i) in activated T cells: (1)Fluo-3–loaded T cells were activated by cocentrifugationwith peptide-loaded APC and the kinetics of fluorescenceintensity changes was monitored continuously and (2)peptide-loaded APC and T cells were mixed, cocultured,and the fluorescence intensity was measured at varioustime intervals.Results: The calcium signal of T cells was dependent onthe APC as demonstrated by the ratio of cells exhibitinghigh versus low fluorescence intensity and by the magnitudeof the calcium signal in the activated population.Short-term interaction of T cells with less potent APC orwith efficient APC in the presence of low antigen concentrationresulted in decreased calcium signaling. CD28-mediated costimulation enhanced the magnitude and sustainedthe increase of intracellular calcium levels. In linewith the strong and sustained calcium signals, the activationof the calcium-dependent transcription factors NF-AT,AP-1, and NF-B was induced.Conclusions: Flow cytometric methods, feasible for therapid and flexible analysis of calcium signaling upon antigen-specificT-cell activation, were established. Kinetics ofthe increase of mean fluorescence intensity reflected thecalcium response of the total cell population whereasstatistical analysis of fluorescence intensity at selectedtime points provided information on the activation state ofsingle cells. Cytometry 47:207–216, 2002

Autologous apoptotic neutrophils inhibit inflammatory cytokine secretion by human dendritic cells, but enhance Th1 responses

Neutrophils represent the most abundant cell type in peripheral blood and exhibit a remarkably brief (6-8 h) half-life in circulation. The fundamental role of these professional phagocytes has been established in acute inflammation, based on their potential to both initiate and receive inflammatory signals. Furthermore, neutrophils also take part in maintaining chronic inflammatory processes, such as in various autoimmune diseases. Here, we demonstrate that human autologous apoptotic neutrophils are readily engulfed by immature monocyte-derived dendritic cells (moDCs) with similar efficiency as allogeneic apoptotic neutrophils [Majai G et al. (2010) J Leukoc Biol 88, 981-991]. Interestingly, in contrast to the allogeneic system, exposure of moDCs to autologous apoptotic neutrophils inhibits LPS + IFN-γ-induced production of inflammatory cytokines in a phagocytosis-independent manner. Autologous apoptotic neutrophil-primed DCs are able to modulate T-cell responses by inducing the generation of IFN-γ-secreting cells while hampering that of IL-17A-producing cells. Our observations indicate that capture of autologous apoptotic neutrophils by immature DCs may impede further neutrophil-mediated phagocytosis and tissue damage, and allow increased clearance of dying cells by macrophages.L

IL-7 withdrawal induces a stress pathway activating p38 and Jun N-terminal kinases

IL-7 delivers survival signals to cells at an early stage in lymphoid development. In the absence of IL-7, pro-T cells undergo programmed cell death, which has previously been associated with a decline in Bcl-2 and translocation of Bax from cytosol to mitochondria. A new, earlier feature of IL-7 withdrawal was identified using an IL-7-dependent thymocyte line. We observed that withdrawal of IL-7 induced increased expression of jun and fos family member genes including c-jun, junB, junD, c-fos and fra2. This transient response peaked 3-4 h after IL-7 was withdrawn and resulted in increased DNA-binding activity of AP-1 and in a change in the composition of the Jun/Fos family dimers shown by electrophoretic mobility shift and supershift assays. Induction of jun and fos genes and the increased DNA-binding activity of AP-1 were attributable to the phosphorylation-induced activation of the stress kinases p38 and JNK and were blocked by the chemical kinase inhibitors SB203580 and SB202190. The stress response contributed to cell death following IL-7 withdrawal as shown by blocking the activity of the stress (MAP) kinases or by blocking the production of c-Jun and c-Fos using antisense oligonucleotides