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First published online 13 June 2007
doi: 10.1242/jcs.007203

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Ca²⁺ signaling through ryanodine receptor 1 enhances maturation and activation of human dendritic cells

Laura Bracci^1,*,, Mirko Vukcevic^3,*, Giulio Spagnoli¹, Sylvie Ducreux², Francesco Zorzato³ and Susan Treves^2,

¹ Institute of Surgical Research, Basel University Hospital, Hebelstrasse 20, 4031 Basel, Switzerland
² Departments of Anesthesia and Research, Basel University Hospital, Hebelstrasse 20, 4031 Basel, Switzerland
³ Department of Experimental and Diagnostic Medicine, General Pathology Section, University of Ferrara, 44100 Ferrara, Italy

View larger version (31K): [in this window] [in a new window]   Fig. 1. Expression of RyR in human DCs. Peripheral blood monocytes were induced to differentiate into iDCs by 5-day culture in IL4 and GM-CSF. (A) Cells were then washed and incubated in the presence of fluorochrome-labeled monoclonal antibodies (mAbs) recognizing the indicated surface markers. Specific fluorescence (full histograms) was evaluated by taking advantage of a FACSCalibur flow cytometer equipped with Cell Quest software (Becton Dickinson) using, as negative controls, isotype-matched irrelevant reagents (empty histograms). One representative experiment out of five is shown. (B) Total RNA was extracted from immature (iDCs), LPS-matured (mature DCs) and from other cell types, and the expression of genes encoding the different RYR isoforms was evaluated by RT-PCR. RNA from EBV-transformed lymphocytes was used as control for RYR1 isoform gene expression. (C) Immunofluorescence analysis of iDCs. Cells were stained with goat anti-RyR polyclonal Ab followed by Alexa Fluor-555-conjugated anti-goat Ab (a-j), Alexa Fluor-555-conjugated anti-goat Ab alone (k) or with allophycocyanine-conjugated anti-CD1a (l). Immunofluorescence analysis on acetone:methanol-fixed iDCs reveals strong perinuclear RyR fluorescence that extends into the endoplasmic reticulum network. Panels a-j show 1 µm optical slices from the bottom upwards; bar, 10 µm. Images were acquired with a 100x Plan Neofluar oil immersion objective (NA 1.3) mounted on a Zeiss Axiovert 100 confocal microscope. Panel l: paraformaldehyde fixed DCs display typical plasma membrane fluorescence for the CD1a marker (bar, 20 µm).

View larger version (32K): [in this window] [in a new window]   Fig. 2. Single-cell intracellular calcium imaging on iDCs. (A,C) Single-cell intracellular calcium measurements in fura-2-loaded iDCs obtained after 5 days of culture. The traces show changes in fura-2 fluorescence ratio (340/380 nm) in a single iDC after addition of caffeine or ATP. Experiments were performed in Krebs-Ringer containing 1 mM Ca2+. (B,D) Average peak increases in fluorescence ratio induced by the addition of the indicated agonists to fura-2-loaded iDCs. Results represent the mean increase in fluorescence (calculated by subtracting the peak fluorescent ratio from the resting fluorescent ratio) obtained after addition of 600 µM 4-chloro-m-cresol, 10 mM caffeine or 150 mM KCl (B) or 100 µM ATP (D). No difference was found in the peak Ca2+ in response to RyR activation (P=0.820), but the amount of Ca2+ released by 100 µM ATP was significantly higher (P<0.00001). Results represent the mean (± s.e.m.) increase in fluorescence in DCs isolated from four different donors.

View larger version (27K): [in this window] [in a new window]   Fig. 3. RyR activation induces transcription of genes involved in DC maturation and potentiates allospecific T-cell stimulation. (A) In vitro-derived iDCs were cultured for 18 hours in the presence of the indicated concentrations of LPS and with 10 mM caffeine and 20 µM dantrolene, as indicated. Total RNA was extracted and CD83, IFN, IL12B and IL23A gene expression was evaluated by quantitative real-time PCR. Gene expression results are expressed as fold-increase as compared with values obtained in iDCs treated with medium + 1 ng/ml LPS. One representative experiment out of four is shown. (B) Experiment as in A except that in vitro-derived iDCs were cultured for 18 hours in the presence of 1 ng/ml LPS + 100 µM ATP (left panel) or in the presence of 10 mM caffeine and 20 µM dantrolene, as indicated (right panel). Gene expression results are expressed as fold-increase as compared with values obtained in iDCs treated with medium + 1 ng/ml LPS (left panel; ATP experiments) or as fold-increase as compared with values obtained in iDCs treated with medium alone (right panel). One representative experiment out of four is shown. (C) Experiment as in A except that in vitro-derived iDCs were cultured for 18 hours in the presence of 1 µM thapsigargin, or the indicated concentration of caffeine or 1 µM thapsigargin + 1 ng/ml LPS. Total RNA was extracted and IL23A gene expression was evaluated by quantitative real-time PCR. One representative experiment out of four is shown. (D) Immature DCs were harvested on day 5 of differentiation and stimulated with 1 ng/ml LPS (grey box), with 10 mM caffeine + 1 ng/ml LPS (hatched box), with 20 µM dantrolene + 10 mM caffeine + 1 ng/ml LPS (diagonal lines) or with 100 µM ATP + 1 ng/ml LPS (horizontal lines), or left untreated (empty box). DCs were then washed and added to allogenic PBMC at a ratio of 10:1 for 5 days. [3H] thymidine was then added and cells were cultured for another day. The bars indicate the mean value of c.p.m. (± s.e.m.) from triplicate samples from one donor. The experiment was repeated with similar results at least four times with different donors.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Incubation of iDCs with KCl or soluble extracts from necrotic cells promotes surface expression of CD83. iDCs were treated for 4 hours as indicated and the percent positive CD83 cells was determined by flow cytometry. (A) Stimulation with 10 mM caffeine alone or 100 µM ATP + 1 ng/ml LPS did not significantly increase CD83 surface expression; incubation with 10 mM caffeine + 1 ng/ml LPS significantly increased CD83 surface expression (P<0.001). This effect was blocked by pretreatment with 20 µM dantrolene. (B) When KCl was added, cells were incubated with Krebs-Ringer saline (in which the NaCl had been substituted for KCl) for 30 minutes at 37°C; they were then centrifuged, and fresh medium containing 1 ng/ml LPS was added and cells were incubated at 37°C for 4 hours. (C) For experiments in which iDCs were incubated with the supernatant from necrotic HEK293 cells, extracts were prepared as described in the Materials and methods section and either used directly (+ necrotic cell extract ± 20 µM dantrolene) or dialysed overnight against 1xPBS (dialysed necrotic cell extract). Results are expressed as mean (± s.e.m.) percentage induction of CD83 surface expression of at least three experiments performed on iDCs purified from blood of different donors; values obtained by treating iDCs with 1 µg/ml LPS was considered 100%. *P and **P, significant difference in the treated population compared with iDCs treated with 1 ng/ml. ***P, significant difference from iDCs treated with 1 ng/ml LPS + necrotic cell extracts.

View larger version (26K): [in this window] [in a new window]   Fig. 5. LPS but not caffeine induces nuclear translocation of NF-kB, whereas caffeine-induced maturation is sensitive to cyclosporine A (CsA). iDCs were left untreated or stimulated for 5 or 60 minutes at 37°C, as indicated. Cells were allowed to stick to poly-L-lysine-treated coverslips and permeabilized with acetone:methanol. Cells were incubated with rabbit anti-p65 polyclonal antibodies (Ab), followed by Alexa Fluor-488-labeled secondary Ab and visualized with a confocal microscope as indicated in the Materials and Methods section. Arrowheads indicate nuclear fluorescence. Images were acquired with a 100x Plan Neofluar oil immersion objective (NA 1.3) mounted on a Zeiss Axiovert 100 confocal microscope. Horizontal bar, 10 µm. Arrowheads point to nuclear translocation of p65. (B) Surface expression of CD83 in unstimulated iDCs (empty bars) or iDCs stimulated with 1 ng/ml LPS (dark-grey bars) ± 10 mM caffeine (light-grey bars) or pretreated with 2 µM CsA for 30 minutes and then with 10 mM caffeine + 1 ng/ml LPS (slashed grey bars), or pretreated with 10 µM deltamethrin for 30 minutes and then with 10 mM caffeine + 1 ng/ml LPS (hatched grey bars). Results are expressed as percent (mean ± s.e.m.; n=3) induction of CD83 surface expression: 100% was the value obtained by stimulating cells with 1 µg/ml LPS for 4 hours. **P, significant difference in the treated population compared with iDCs treated with 1 ng/ml.

View larger version (28K): [in this window] [in a new window]   Fig. 6. Cartoon depicting signaling pathways involved in DC maturation. In the presence of sub-optimal Toll-like receptor ligand, the signal(s) generated from cells dying in the vicinity of iDCs bind to receptors that are coupled to the RyR1, activating calcium release. This intracellular pathway leading to DC maturation through RyR1 activation is dependent upon the activity of calcineurin as it could be blocked by cyclosporine A and deltamethrin. PM, plasma membrane.

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