Celecoxib-induced cell death in rheumatoid arthritis fibroblast-like synoviocytes is caspase-independent

<p><b>Copyright information:</b></p><p>Taken from "Apoptosis is not the major death mechanism induced by celecoxib on rheumatoid arthritis synovial fibroblasts"</p><p>Arthritis Research & Therapy 2007;9(6):R128-R128.</p><p>Published online 12 Dec 2007</p><p>PMCID:PMC2246250.</p><p></p> Effect of caspase inhibition on celecoxib-induced cell death. Cells were pre-treated with caspase inhibitors (pancasp-In: pan-caspase inhibitor z-VAD-fmk; casp3-In: caspase 3 inhibitor z-DEVD-fmk) or control inhibitor z-FA-fmk (co-In: control inhibitor) for 1 hour and subsequently cultured in the presence of either 60 μM celecoxib or 0.5 nM tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) for an additional 24 hours. Cells pre-treated only with solvent (dimethyl sulfoxide [DMSO]) served as controls. Cell death was determined using Annexin V binding and TO-PRO-3 uptake and expressed as relative cell death. (For this, cell death induced by TRAIL or celecoxib plus inhibitor was first subtracted by cell death of cells treated with inhibitor alone and then expressed as percentage versus cell death induced by TRAIL or celecoxib alone.) Data from three patients were averaged and are shown as the mean ± standard error of the mean

Characterization of celecoxib-induced cell death in rheumatoid arthritis fibroblast-like synoviocytes

<p><b>Copyright information:</b></p><p>Taken from "Apoptosis is not the major death mechanism induced by celecoxib on rheumatoid arthritis synovial fibroblasts"</p><p>Arthritis Research & Therapy 2007;9(6):R128-R128.</p><p>Published online 12 Dec 2007</p><p>PMCID:PMC2246250.</p><p></p> Cells were treated at indicated concentrations and times with celecoxib or staurosporine or tumor necrosis factor-related apoptosis-inducing ligand. Apoptosis was evaluated by fluorescence-activated cell sorting analysis using Annexin V binding and TO-PRO-3 uptake. Representative data of three different experiments are shown. DMSO, dimethyl sulfoxide

Comparison of morphological changes of rheumatoid arthritis fibroblast-like synoviocytes (RA FLSs) treated with either celecoxib or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by light microscopy (magnification × 300)

<p><b>Copyright information:</b></p><p>Taken from "Apoptosis is not the major death mechanism induced by celecoxib on rheumatoid arthritis synovial fibroblasts"</p><p>Arthritis Research & Therapy 2007;9(6):R128-R128.</p><p>Published online 12 Dec 2007</p><p>PMCID:PMC2246250.</p><p></p> Untreated RA FLSs (upper panel) and cells treated for either 1 hour with 60 μm celecoxib (middle panel) or 8 hours with 1 nM TRAIL (lower panel) are shown. Apoptotic cells are indicated by arrows

Celecoxib induces cell death in rheumatoid arthritis fibroblast-like synoviocytes

<p><b>Copyright information:</b></p><p>Taken from "Apoptosis is not the major death mechanism induced by celecoxib on rheumatoid arthritis synovial fibroblasts"</p><p>Arthritis Research & Therapy 2007;9(6):R128-R128.</p><p>Published online 12 Dec 2007</p><p>PMCID:PMC2246250.</p><p></p> Cells were treated at indicated concentrations and times with either celecoxib or staurosporine. Apoptosis was evaluated by fluorescence-activated cell sorting analysis using Annexin V binding and TO-PRO-3 uptake. Values are expressed as the percentage of total cell death (upper panel) or apoptosis (lower panel) and are the mean ± standard error of the mean of three individual experiments

Celecoxib does not induce caspase activation in rheumatoid arthritis fibroblast-like synoviocytes (RA FLSs)

<p><b>Copyright information:</b></p><p>Taken from "Apoptosis is not the major death mechanism induced by celecoxib on rheumatoid arthritis synovial fibroblasts"</p><p>Arthritis Research & Therapy 2007;9(6):R128-R128.</p><p>Published online 12 Dec 2007</p><p>PMCID:PMC2246250.</p><p></p> FLSs were stimulated for 2 hours with celecoxib at indicated concentrations or for 4 hours with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (0.5 nM) as positive control. Cell lysates were analyzed by immunoblot for caspase 3 expression. The same blot was stripped and reprobed with a mouse anti-human β-actin antibody to confirm equal loading. One representative immunoblot is shown. RA FLSs were stimulated for indicated time points with 60 μM celecoxib or with TRAIL (0.5 nM) as positive control, and caspase 3 activity was measured using Ac-DEVD-AMC protease assay. Caspase 3 activity is expressed as fold increase to unstimulated cells (NS) and is represented as the mean ± standard error of the mean (SEM) of different experiments using RA FLSs from three different patients. RA FLSs were stimulated for indicated time points with celecoxib at indicated concentrations or with TRAIL (0.5 nM) as positive control. Cell lysates were analyzed by immunoblot for poly(ADP-ribose) polymerase (PARP) and caspase 8 and 9 expression. One representative immunoblot is shown. RA FLSs were stimulated for 12 hours with celecoxib at indicated concentrations or with TRAIL (0.5 nM) as positive control, and DNA fragmentation was measured using the Cell Death Detection ELISAkit. The enrichment of mono- and oligonucleosomes released into the cytoplasm is calculated as the ratio of the absorbance of the sample cells to the absorbance of control cells and is shown as the mean ± SEM from three experiments performed in duplicate

Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells

<div><p>B cells can have a regulatory role, mainly mediated by interleukin 10 (IL-10). IL-10 producing B cells (B10 cells) cells remain to be better characterized. Annexin V binds phosphatidylserine (PS), which is externalized during apoptosis. Previous works suggested that B10 cells are apoptotic cells since they bind Annexin V. Others showed that Annexin V binding could also be expressed on viable B cells. We aimed to explore if PS exposure can be a marker of B10 cells and if PS exposure has a functional role on B cell IL-10 production in healthy subjects. We found that B10 cells were significantly more often Annexin V⁺ than IL-10 non-producing B cells. After CpG activation, Annexin V⁺ B cells differentiated more often into B10 cells than Annexin V^neg B cells. Cell death and early apoptosis were similar between Annexin V⁺ and Annexin V^neg B cells. PS blockage, using biotinylated AnV and glyburide, decreased B10 cell differentiation. This study showed that B10 cells have an increased PS exposure independently of any apoptotic state. B cells exposing PS differentiate more into B10 cells whereas PS blockage inhibits B10 cells generation. These results strongly suggest a link between PS exposure and B10 cells.</p></div

Annexin V positive B cells are not apoptotic cells.

<p>PBMCs were stained with APC anti-CD19 and FITC conjugated Annexin V, sorted by FACSARIA according to annexin V (AnV) staining: high AnV B cells (AnV^hi); low AnV B cells (AnV^low) and AnV negative B cells (AnV^neg). Cell death was analyzed at 72 hr using DAPI staining for necrosis (A) and DIOC6 (B) for mitochondrial apoptosis (n = 4). Additionally, cell cycle was analyzed using propidium iodide (PI) staining at 72 hr (C), showing the proportion of cells in the subG1 phase (apoptotic cells) and in the S phase (proliferating cells) in AnV+ (AnV^hi and AnV^low) and AnV^neg B cells (n = 5). Data are median (IQR25-75).</p

Phosphatidylserine blockage alters B10 cell differentiation.

<p>PBMCs from 6 healthy subjects were pretreated with biotinylated Annexin V (biot AnV) (n = 8) or with glyburide (Gly) (n = 8) and then stimulated for 24 hr according to the protocol for B10 assessment previously described. A shows representative plots and B shows the results as median (IQR25-75).</p

Annexin V binding is increased among CD5+ and CD24hiCD27+ B10 precursors.

<p>A representative plot is presented (A). PBMCs from 12 subjects were analyzed by FACS for B10 precursors phenotype (i.e CD19+CD5+ (B), CD19+CD24^hiCD27⁺ (C) and CD19+CD24^hiCD38^hi, (D)), and for annexin V binding. Wilcoxon’s matched pairs signed rank tests were used. Data are median (IQR 25–75).</p

Annexin V binding is increased in IL-10 producing B cells (B10 cells) compared to IL-10 non-producing B cells and to TNFα producing B cells.

<p>PBMCs were stimulated for 24 hr with CpG/ionomycin/PMA and BFA as described previously and analyzed by FACS for annexin V (AnV) binding and IL-10 and TNFα production in 21 subjects. Cytometry gating is shown in A. Results are presented in percentage of positive cells in B and median of fluorescence (MFI) in C. Wilcoxon’s matched pairs signed rank tests were used. Top of the bar represents the median, and whiskers are IQR 25–75.</p