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Phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase regulate induction of Mcl-1 and survival in glucocorticoid-treated human neutrophils 

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Presentation on theme: "Phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase regulate induction of Mcl-1 and survival in glucocorticoid-treated human neutrophils "— Presentation transcript:

1 Phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase regulate induction of Mcl-1 and survival in glucocorticoid-treated human neutrophils  Arash S. Saffar, DDS, Stéphane Dragon, BSc, Peyman Ezzati, BSc, Lianyu Shan, MSc, Abdelilah Soussi Gounni, PhD  Journal of Allergy and Clinical Immunology  Volume 121, Issue 2, Pages e10 (February 2008) DOI: /j.jaci Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Dexamethasone upregulated antiapoptotic Mcl-1. Freshly isolated neutrophils (time 0) and those cultured for various time points were analyzed for Mcl-1 by means of immunoblotting and densitometry (A and B) or real-time RT-PCR (C). ∗P < .05; ∗∗P < .01 (n = 6). Dex, Dexamethasone; RU, RU-486; MG, MG-132; Med, medium. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Inhibition of PI3K and p38 MAPK suppressed glucocorticoid-mediated survival of neutrophils. Freshly isolated human neutrophils were preincubated with 25 nmol/L wortmannin (A), 5 μmol/L U0126 (B), 10 μmol/L SP (C), 25 μmol/L SB (D), or dimethyl sulfoxide before stimulation with dexamethasone (Dex), GM-CSF, or medium alone. Apoptosis was assessed by using annexin-V/propidium iodide staining. ∗∗∗P < .0005; ∗∗P < .005 (n = 8-9). Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 PI3K and p38 MAPK regulated the induction of survival and Mcl-1 by dexamethasone. Neutrophils were assessed for apoptosis by using annexin-V/propidium iodide staining (A and B; 1 μmol/L U0126, 10 μmol/L SB203580, or 10 nmol/L wortmannin), immunoblotted for Mcl-1 (C; 25 μmol/L SB or 25 nmol/L wortmannin), and analyzed after 18 hours for mitochondrial potential or after 6 hours for caspase-3 activity (D and E; 25 μmol/L SB203580, 25 nmol/L wortmannin, or 10 μmol/L SB plus 10 nmol/L wortmannin). ∗∗∗P < .001; ∗∗P < .01; ∗P < .05. SB, SB203580; Dex, dexamethasone; Wort, wortmannin; RU, RU-486. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Dexamethasone delayed apoptosis of neutrophils
Dexamethasone delayed apoptosis of neutrophils. A, Freshly isolated human neutrophils were cultured and assessed for apoptosis by means of annexin-V/propidium iodide staining (∗∗∗P < and ∗∗P < .005 compared with medium; n = 8). B, Freshly isolated human neutrophils were cultured for 24 hours and assessed for apoptosis by using a DNA fragmentation assay (∗∗∗P < and ∗∗P < .01 compared with medium; n = 5). Dex, Dexamethasone; RU, RU-486; CHX, 10 ng/mL cycloheximide. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Glucocorticoids induced neutrophil survival independently of transrepression. Freshly isolated human neutrophils were cultured with various concentrations of CpdA and assessed for apoptosis by using either annexin-V/propidium iodide staining (A; ∗∗∗P < compared with medium; n = 4) or DNA fragmentation assay (B; 24-hour culture; ∗∗P < .005 compared with medium; n = 3). C, Expression of Fas was analyzed by means of FACS in neutrophils cultured for 18 hours. The shaded line represents the isotype control. The average of mean fluorescence intensity (MFI) was calculated from 3 independent experiments. D, Freshly isolated neutrophils (time 0) and those cultured for 6 and 12 hours were lysed and analyzed for total levels of Bax and Bid by means of immunoblotting. D represents 3 separate experiments. Dex, Dexamethasone; RU, RU-486. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Dexamethasone upregulated antiapoptotic XIAP protein
Dexamethasone upregulated antiapoptotic XIAP protein. IAPs were detected in total lysates of fresh neutrophils or neutrophils cultured for 6 and 12 hours by means of Western blotting and quantified by means of densitometry. Densitometric results are reported as fold increase over average value for time 0. The figure represents 3 independent experiments (∗P < .05; ∗∗P < .01). Dex, Dexamethasone; RU, RU-486; XIAP, X-chromosome linked inhibitor of apoptosis protein; cIAP, cellular inhibitor of apoptosis protein. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Dexamethasone suppressed proapoptotic activity at the level of mitochondria. Western blotting was performed on subcellular fractions of freshly isolated neutrophils (time 0) or those cultured for 18 hours. Manganese superoxide dismutase (MnSOD) and GAPDH served as mitochondrial and cytosolic content controls, respectively. Panels represent 3 separate experiments. Dex, Dexamethasone; RU, RU-486. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Caspase activity in neutrophils was suppressed by dexamethasone
Caspase activity in neutrophils was suppressed by dexamethasone. Enzymatic activity of caspase-8 and caspase-9 (A) and caspase-3 (B) was measured in neutrophils cultured for various time points. Data are reported as fold increase over average value for GM-CSF. Anti-Fas IgM (250 ng/mL) served as a positive control (∗P < .05; ∗∗∗P < .001; n = 5, performed in triplicate). C, Western blots were performed for active caspase-3 and its zymogen (procaspase-3) on lysates from fresh neutrophils (time 0) or those cultured for 12 and 18 hours. C represents 3 separate experiments. Dex, Dexamethasone; RU, RU-486. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Inhibition of PI3K blocked dexamethasone-induced survival of neutrophils without causing necrosis. Freshly isolated human neutrophils were preincubated with 25 nmol/L wortmannin or dimethyl sulfoxide before stimulation with dexamethasone or medium alone. Representative FACS plots of annexin-V/propidium iodide staining patterns of neutrophils are shown. Dex, Dexamethasone. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Akt and p38 MAPK were activated after dexamethasone stimulation of neutrophils. Freshly isolated human neutrophils were preincubated with 25 nmol/L wortmannin (A), 25 μmol/L SB (B), or dimethyl sulfoxide before stimulation with dexamethasone or medium alone. These cells were collected and lysed at the specified time points, followed by immunoblotting for total and phosphorylated Akt (Fig E7, A) or p38 MAPK (Fig E7, B). The figure is representative of 5 separate experiments. Dex, Dexamethasone. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Glucocorticoid-induced survival of human neutrophils: a schematic model. After treatment of neutrophils with dexamethasone, dimerization and movement of the glucocorticoid receptor to the nucleus is thought to occur. Within hours, Mcl-1 and IAPs are upregulated, correlating with maintenance of mitochondrial integrity and suppression of caspases. PI3K and p38 MAPK regulate this process at or upstream of mitochondria. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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