Figures
Figure 1
Experimental strategy.
Initially, primaryBMDCs and BMMFs were evaluated for their capacity to take up conalbumin. Then, a stable in vitro co-culture model of T lymphocytes and APCs was generated to study cell growth and cytokine secretion in T lymphocytes as the result of antigen presentation. (Illustration was made on the basis of Servier Medical Art).
Figure 1
Figure 2
Characterization of primary BMDCs and BMMFs.
After isolation, the cells were cultured for 10 days as described in the Method.
A) The morphology of the cells was examined by phase-contrast microscopy (magnification 20×). Left panel: BMDCs; right panel: BMMFs. B) BMDCs (left panel) or BMMFs (right panel) were incubated with fluorescein isothiocyanate-conjugated ovalbumin (FITC-OVA) as described. Fluorescence intensity was analyzed by FACS.
Figure 2
Figure 3
The effect of different concentrations of extracellular Mg and Ca blockers on endocytosis by BMDCs and BMMFs. BMDCs (A) and BMMFs (B) were cultured in 0.5, 1.0 or 5.0 mM Mg containing medium with or without verapamil or/and TMB-8 (both 25 μM). After pre-incubation with FITC-OVA for 6 h, at 37°C, fluorescence intensity was analyzed by FACS. Results are expressed as the mean ± SD of five separate experiments. Different letters indicate significant effect (p<0.05) of treatments with Ca blockers within the same magnesium concentration used (a, b, c).
Figure 3
Figure 4
The effect of different concentrations of extracellular Mg and Ca blockers on the proliferation of D10.G4.1 lymphocytes co-cultured with BMDCs or BMMFs. BMDCs and BMMFs were cultured in 0.5, 1.0 or 5.0 mM Mg containing medium with or without verapamil (ver) or/and TMB-8 (both 25 μM) for 72 h. A, C) cell proliferation assay on BMDCs and BMMFs, respectively. B, D) MTT assay onBMDCs and BMMFs, respectively.
The values are the mean ± SD from five independent experiments. Different letters indicate significant effect (p<0.05) of treatments: (a, b) effect of various Mg concentrations; (w, x, y, z) effect of Ca on cells cultured in 1 0 mM Mg.
Figure 4
Figure 5
The effect of different concentrations of extracellular Mg and Ca blockers on IL-4 secretion by D10.G4.1 lymphocytes co-cultured with BMDCs or BMMFs.
BMDCs (A) and BMMFs (B) were cultured in 0 5, 1.0 or 5.0 mM Mg containing media with or without verapamil or/and TMB-8 (both 25 μM) for 24 h. IL-4 levels were measured by ELISA.
The values are the mean ± SD from five independent experiments. Different letters indicate significant effect (p<0.05) of treatments: (a, b) effect of various Mg concentrations; (w, x, y, z, v, q) effect of Ca blockers on cells cultured in 1.0 mM Mg concentration.
Figure 5
Figure 6
The effect of different concentrations of extracellular Mg and Ca blockers on IL-10 secretion by D10.G4.1 lymphocytes co-cultured with BMDCs or BMMFs.
BMDCs (A) and BMMFs (B) were cultured in 0 5, 1.0 or 5.0 mM Mg with or without verapamil or/and TMB-8 (both 25μM) for 24 h. IL-10 levels were measured by ELISA.
The values are the mean ± SD from five independent experiments. Different letters indicate significant effect (p<0.05) of treatments: (a, b) effect of various Mg concentrations; (w, x, y, z, v, q) effect of Ca blockers on cells cultured in the control Mg concentration (1.0 mM).
Figure 6
Tables
Authors
1 Department of Immunology, Pathophysiology and Veterinary Preventive Medicine, Wroclaw University of Environmental and Life Science, C.K. Norwida 31, 50-375 Wroclaw, Poland
Recognition of a pathogen is a fundamental function of the immune system. Important players in this process are the so-called antigen-presenting cells (APCs) which engulf, process, and then present antigens to T lymphocytes. APCs also produce the signals necessary for the activation of lymphocytes into effector cells that actively carry out immune defenses. In addition, they prime self-reactive T cells [1]. APCs are a heterogeneous population that includes Langerhans cells, macrophages, dendritic [...]