The unspecific effect of PP on thymocyte maturation is not reversed by exogenous AA and PGE₂.

<p><b>A.</b> Representative thymocyte subpopulation distribution in WT cPLA₂α FTOC after 5 days of culture in absence or presence of 1μM of PP and exogenous (1μM) AA and PGE₂. Thymocytes were identified cytofluorometrically using fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8. <b>B.</b> WT cPLA₂α fetal thymuses were cultured during 5 days as FTOCs in absence or presence of 1μM of PP, and exogenous (1μM) AA and PGE₂. After mechanical dissociation of fetal thymuses, the thymocytes were identified with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 3 independent experiments and the number of fetal thymuses for each condition is: Diluent (n = 5); 1μM PP (n = 6); 1μM PP and 1μM AA (n = 5); 1μM PP and 1μM PGE₂ (n = 5). * P< .05; ** P< .01; *** P< .001.</p

Eicosanoid profiles of cPLA₂α WT and KO FTOC supernatants and adult mouse thymuses.

<p><b>A.</b> Expression distribution of the eicosanoids present in cPLA₂α WT FTOCs. The supernatants of FTOCs were collected at the indicated time of culture and the eicosanoid profiles were determined by combined liquid chromatography/tandem mass spectrometry. Data are mean of 3 different supernatants. <b>B.</b> Eicosanoid profiles of cPLA₂α WT and KO FTOC supernatants. The supernatants of FTOCs were collected at the indicated time of culture and eicosanoid profiles were determined by combined liquid chromatography/tandem mass spectrometry. Data are mean ± SEM of 3 different supernatants. <b>C.</b> Eicosanoid profiles of cPLA₂α WT and KO thymuses from adult mice. Adult thymuses were mechanically disrupted and eicosanoid profiles were determined by combined liquid chromatography/tandem mass spectrometry. ** P< .01, data are means ± SEM of 3 cPLA₂α WT thymuses and 2 cPLA₂α KO thymuses.</p

cPLA₂α inhibition does not impact human thymocyte maturation.

<p><b>A.</b> Representative thymocyte subpopulation distribution in human FTOC after 5 days of culture in absence or presence of indicated concentrations of PP. Thymocytes were identified cytofluorometrically using fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8. <b>B.</b> Human FTOCs were cultured during 5 days in absence or presence of indicated concentrations of PP. After mechanical dissociation of human FTOCs, the thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 3 independent experiments and the number of thymuses for each condition is: Diluent (n = 6); 10nM PP (n = 6); 100nM PP (n = 6); 1000nM PP (n = 6). NS (non significant).</p

The disruption of the cPLA₂α gene does not impact thymocyte maturation in FTOC.

<p><b>A.</b> Representative thymocyte subpopulation distribution in WT and KO cPLA₂α FTOC. After 5 days of culture, the identification of thymocytes with fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8 was determined by flow cytometry. <b>B.</b> WT and KO cPLA₂α fetal thymuses were cultured during 5 days as FTOCs. After mechanical dissociation of fetal thymuses, the thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8, and analyzed by flow cytometry. Data are mean ± SEM of 6 independent experiments and the number of fetal thymuses for each genotype is: cPLA₂^+/+ (n = 17); cPLA₂^-/- (n = 9). NS (non significant).</p

Study of the Role of Cytosolic Phospholipase A₂ Alpha in Eicosanoid Generation and Thymocyte Maturation in the Thymus

<div><p>The thymus is a primary lymphoid organ, home of maturation and selection of thymocytes for generation of functional T-cells. Multiple factors are involved throughout the different stages of the maturation process to tightly regulate T-cell production. The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses. In this study, we determined the profile of expression of the eicosanoids present in the mouse thymus at different stages of thymocyte development. As the group IVA cytosolic phospholipase A₂ (cPLA₂α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA₂α deficient mice to our investigations. We found that a vast array of eicosanoids is expressed in the thymus, which expression is substantially modulated through thymocyte development. The cPLA₂α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA₂α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA₂α inhibitor pyrrophenone did not impact thymocyte maturation. This study provides information on the eicosanoid repertoire present during thymocyte development and suggests that thymocyte maturation can occur independently of cPLA₂α.</p></div

Pharmacological blockade of cPLA₂α does not affect thymocyte maturation in FTOC.

<p><b>A.</b> Representative thymocyte subpopulation distribution in WT cPLA₂α FTOC after 5 days of culture in absence or presence of indicated concentrations of PP. Thymocytes were identified with fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8 by flow cytometry. <b>B.</b> WT cPLA₂α fetal thymuses were cultured during 5 days as FTOCs in absence or presence of indicated concentrations of PP. After mechanical dissociation of fetal thymuses, the thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 5 independent experiments and the number of fetal thymuses for each condition is: Diluent (n = 10); 10nM PP (n = 8); 100nM PP (n = 12). NS (non significant).</p

cPLA₂α inhibition by high concentration of PP impacts thymocyte maturation in FTOC.

<p><b>A.</b> Representative thymocyte subpopulation distribution in WT cPLA₂α FTOC after 5 days of culture in absence or presence of 1μM of PP. Thymocytes were identified cytofluorometrically using fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8. <b>B.</b> WT cPLA₂α fetal thymuses were cultured during 5 days as FTOCs in absence or presence of 1μM of PP. After mechanical dissociation of fetal thymuses, the thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 9 independent experiments and the number of fetal thymuses for each condition is: Diluent (n = 22); 1μM PP (n = 13). *** P< .001.</p

Study of swarm detection in high sensitivity flow cytometry.

<p>(A) A mixture of CMFDA^- and CMFDA⁺ platelet MPs (CMFDA^{- and +}) and sky blue beads (220 nm in diameter) were analyzed alone (left and middle panel respectively) or mixed (right panel) and their detection resolved on the basis of fluorescence. (B) CMFDA^{- and +} platelet MPs and sky blue beads (450 nm in diameter) were analyzed alone (left and middle panel respectively) or mixed (right panel) prior to detection on the basis of fluorescence. (C) CMFDA⁺ platelet MPs and RBC MPs labeled with antibodies directed against TER 119 are analyzed alone (left and middle panel respectively) or mixed (right panel). (D, E, F) CMFDA⁺ platelet MPs were diluted serially thrice (2-fold dilution) and analyzed by high sensitivity flow cytometry to determine their concentration (D), the CMFDA-height (H) mean of fluorescence (E) and the CMFDA-H median of fluorescence (F) are presented. Data are mean ± SEM of 5 independent experiments. BKD = Background noise.</p

Optimization of flow cytometric methods for the detection of MPs.

<p>(A, B) Acquisition of fluorescent microspheres of 100nm (Blue), 450nm (pink), 840nm (green), 1000nm (red), 3200nm (orange) in diameter on a flow cytometer Canto II modified with a FSC-PMT small particles option. (B) A MP gate including particles from 100 to 1000nm in diameter based on the microsphere sizes (FSC-PMT-H) is presented and used to detect MPs. (C) Portrayal of relative size of human platelets detected with fluorochrome-conjugated antibodies directed against CD41. (D) FSC-PMT/SSC portrayal of platelet MPs detected with annexin-V and fluorochrome-conjugated antibodies directed against CD41 in absence of treatment (control). (E) A known concentration of auto-fluorescent polystyrene microspheres (15 µm in diameter) was added in each tube and a determined number of beads was acquired in the counting bead gate to quantitatively process the data. (F, G) FSC-PMT/SSC portrayal of platelet MPs detected with annexin-V and fluorochrome-conjugated antibodies directed against CD41 and treated with 0.05% triton (F) and 50µM EDTA (G). Total annexin-V⁺ events are detected in the pink gate (middle panel) and the quantity of annexin-V⁺ MPs is determined in the Annexin-V MP gate (upper panel). Total CD41⁺ events are detected in the blue gate (middle panel) and the quantity of CD41⁺ MPs is determined in the CD41 MP gate (lower panel). Data are representative of 5 independent experiments. (H) Triton sensitivity of the platelet MPs detected using fluorochrome-conjugated annexin-V (left panel) and fluorochrome-conjugated antibodies directed against CD41 (right panel) is presented as % of untreated (control). (I) EDTA sensitivity of annexin-V (left panel) and CD41 (right panel) labeling is presented as % of untreated (control). Data are representative of 5 independent experiments.</p

Impact of human and mouse sPLA₂s on platelet MPs.

<p>(A) MPs from human platelets (stimulated with collagen) labeled with the CMFDA cell tracker were incubated for 1 and 6 hours at 37°c in absence or in presence of indicated concentrations of human recombinant sPLA₂ IIA, V, X, or 1µg/ml of the inactive mutant V H48Q. Fluorochrome-conjugated antibodies directed against CD41 and fluorochrome-conjugated annexin-V were used to assess the quantities of CMFDA⁺ MPs (left panel), of CD41⁺ MPs (middle panel), of annexin-V⁺ MPs (right panel) and were compared to the untreated conditions (dotted line). Data are mean ± SEM of 5 independent experiments presented as % of untreated (control) (B) MPs from mouse platelets (stimulated with collagen), identified using YFP as fluorescent tracker, were incubated 1 and 6 hours at 37°c, in absence or in presence of indicated concentrations of mouse recombinant sPLA₂ IIA, V, X, or 1µg/ml of the inactive mutant X H48Q. Fluorochrome-conjugated antibodies directed against CD41 and fluorochrome-conjugated annexin-V were used to determine the concentrations of YFP⁺ MPs (left panel), of CD41⁺ MPs (middle panel), of annexin-V⁺ MPs (right panel) and then compared to the untreated conditions (dotted line). Data are mean ± SEM of 5 independent experiments presented as % of untreated (control). (C) MPs from human platelets labeled with the CMFDA cell tracker and obtained following stimulation with collagen, thrombin or HA-IgG were incubated 6 hours at 37°c in absence or in presence of indicated concentration of human recombinant sPLA₂ IIA, V and X and 1µg/ml of the inactive mutant sPLA₂ V H48Q. Fluorochrome-conjugated antibodies directed against CD41 and fluorochrome-conjugated annexin-V were used to assess the quantities of CMFDA⁺ MPs (left panel), of CD41⁺ MPs (middle panel), of annexin-V⁺ MPs (right panel) and then compared to the untreated conditions (dotted line). Data are mean ± SEM of 3 independent experiments presented as % of untreated (control). (D) MPs from human platelets (stimulated with collagen) labeled with the CMFDA cell tracker were incubated 6 hours at 37°c in PFP of C57BL6 (supplemented or not with 1µg/ml of recombinant human sPLA₂ IIA) or transgenic mice expressing the human sPLA₂ IIA (Tg). Fluorochrome-conjugated antibodies directed against CD41 and fluorochrome-conjugated annexin-V were used to assess the quantities of CMFDA⁺ MPs (left panel), of CMFDA⁺ CD41⁺ MPs (middle panel) and CMFDA⁺ annexin-V⁺ MPs (right panel). Data are mean ± SEM of 3 independent experiments. (E) Concentrations of Annexin-V⁺ MPs and CD41⁺ MPs present in the synovial fluids of RA patients determined by high sensitivity flow cytometry and correlated to the concentration of human sPLA₂ IIA assayed (in the same synovial fluids) by time-resolved immunofluorescence analysis. * P< .05; # P< .01; § P< .001.</p