Sex-specific alterations in neutrophil apoptosis: the role of estradiol and progesterone by Eleanor J. Molloy, Amanda J. O'Neill, Julie J. Grantham, Margaret Sheridan-Pereira, John M. Fitzpatrick, David W. Webb, and R. William G. Watson Blood Volume 102(7):2653-2659 October 1, 2003 ©2003 by American Society of Hematology

Sex differences in spontaneous neutrophil apoptosis. Sex differences in spontaneous neutrophil apoptosis. Neutrophils (1 × 106 cells/mL) were isolated from healthy male (n = 26) and female (n = 26) volunteers and incubated in vitro for 24 hours. Percentage neutrophil apoptosis was evaluated by propidium iodide DNA staining and flow cytometry. *P < .05 versus men. Error bars indicate mean ± SD. Eleanor J. Molloy et al. Blood 2003;102:2653-2659 ©2003 by American Society of Hematology

Dose-dependent effects of estradiol, progesterone, or both in combination on spontaneous neutrophil apoptosis. Dose-dependent effects of estradiol, progesterone, or both in combination on spontaneous neutrophil apoptosis. Neutrophils (1 × 106 cells/mL) isolated from male (▪; n = 10) and female (♦; n = 10) controls were incubated in vitro with increasing physiologic doses (10-12 to 10-7 g/mL) of estradiol (A), progesterone (B), or both in combination (C). Propidium iodide DNA-binding staining using flow cytometry assessed neutrophil apoptosis at 24 hours. *P < .05 versus control (Con). Error bars indicate mean ± SD. Eleanor J. Molloy et al. Blood 2003;102:2653-2659 ©2003 by American Society of Hematology

Effects of estradiol and progesterone on markers of inflammatory cell activation. Effects of estradiol and progesterone on markers of inflammatory cell activation. Normal neutrophils (1 × 106 cells/mL) were incubated for 6 hours with estradiol, progesterone, or both in combination (10-8 g/mL). Cells were then assessed for CD11b (A) using a PE-labeled mAb and the mean channel fluorescence analyzed using flow cytometry. □ indicates male (n = 8); and ▪ indicates female (n = 8). (B) ROI activity was assessed in neutrophils preincubated with sex steroids for 6 hours and then stimulated with DHR (baseline [BL]) alone or in addition to PMA (respiratory burst [RB]). Results were expressed as the Ln mean channel fluorescence ± SEM. (C) Results were also expressed as the Δ difference of the Ln mean channel fluorescence between DHR-treated cells with and without PMA stimulation. *P < .05 versus control (male, n = 8; female, n = 8). Error bars indicate mean ± SD. Eleanor J. Molloy et al. Blood 2003;102:2653-2659 ©2003 by American Society of Hematology

Effects of estradiol and progesterone on the priming effect. Effects of estradiol and progesterone on the priming effect. Normal neutrophils (1 × 106 cells/mL) were incubated for 1 hour either with or without estradiol, progesterone, or both in combination (10-8 g/mL), before the addition of LPS (1 μg/mL). Cells were then assessed for percentage apoptosis by propidium iodide DNA staining after 24 hours (A) or CD11b expression using a PE-labeled mAb after 6 hours and expressed as Ln mean channel fluorescence (B). □ indicates male (n = 6) and ▪ indicates female controls (n = 6). *P < .05 versus control. Error bars indicate mean ± SD. Eleanor J. Molloy et al. Blood 2003;102:2653-2659 ©2003 by American Society of Hematology

The effects of inhibitors of female sex hormones on neutrophil apoptosis. The effects of inhibitors of female sex hormones on neutrophil apoptosis. Neutrophils (1 × 106 cells/mL) were incubated with estradiol, progesterone, or both (10-7 g/mL) for 24 hours. RU486 (RU; 10-6 M) was added at time 0 hours and percentage apoptosis was assessed by flow cytometry at 24 hours (n = 11). ICI 182 780 (ICI; 10-8 M) was added at time 0 hours and percentage apoptosis was assessed by flow cytometry at 24 hours following propidium iodide staining (n = 8). All experiments used both male and female subjects. *P < .05 versus control; **P < .05 versus estradiol; ***P < 0.05 versus progesterone (mean ± SEM). Eleanor J. Molloy et al. Blood 2003;102:2653-2659 ©2003 by American Society of Hematology

Effects of estradiol and progesterone on Fas-induced apoptosis and Fas receptor expression. Effects of estradiol and progesterone on Fas-induced apoptosis and Fas receptor expression. Neutrophils were incubated with estradiol, progesterone, or both (10-8 g/mL) for 6 hours. (A) Fas antibody was added and the percentage apoptosis assessed at 24 hours by propidium iodide staining and flow cytometry. □ indicates male (n = 10) and ▪ indicates female (n = 10). (B) Neutrophils were incubated with Fas antibody and mean channel fluorescence (LnMCF) was subsequently assessed by flow cytometry. □ indicates male (n = 5) and ▪ indicates female (n = 5). Error bars indicate mean ± SD. Eleanor J. Molloy et al. Blood 2003;102:2653-2659 ©2003 by American Society of Hematology

The effects of estradiol and progesterone on spontaneous caspase 3 activation at 6 and 24 hours assessed by Western blot and densitometry. The effects of estradiol and progesterone on spontaneous caspase 3 activation at 6 and 24 hours assessed by Western blot and densitometry. Total cellular protein was extracted at 6 and 24 hours from control neutrophils and cells incubated with estradiol or progesterone (10-8 g/mL). (A) Pro and active caspase 3 were assessed by Western blot analysis. These blots are representative of one of at least 3 separate experiments. (B) Densitometry was performed and the density of each band (percent of total pixels) expressed as the mean ± SD of all blots. *P < .05 versus control at 6 hours. Eleanor J. Molloy et al. Blood 2003;102:2653-2659 ©2003 by American Society of Hematology

The effects of estradiol and progesterone on spontaneous caspase 9 activation at 24 hours assessed by Western blot and densitometry. The effects of estradiol and progesterone on spontaneous caspase 9 activation at 24 hours assessed by Western blot and densitometry. Total cellular protein was extracted at 24 hours from control neutrophils and cells incubated with estradiol or progesterone (10-8 g/mL). (A) Pro and active caspase 9 were assessed by Western blot analysis. These blots are representative of 1 of at least 3 separate experiments. (B) Densitometry was performed and the density of each band (percent of total pixels) expressed as the mean ± SD of all blots. *P < .05 versus control at 6 hours. Eleanor J. Molloy et al. Blood 2003;102:2653-2659 ©2003 by American Society of Hematology

Spontaneous caspase 3 and 9 activation by estradiol and progesterone at 24 hours using fluorescent caspase substrates. Spontaneous caspase 3 and 9 activation by estradiol and progesterone at 24 hours using fluorescent caspase substrates. Cell lysates were prepared from 10 × 106 cells using caspase isolation and incubation buffers. Aliquots of the lysates were diluted in caspase incubation buffer and Ac-DEVD-AMC (caspase 3; panel A) or Ac-LEHD-AMC (caspase 9; panel B). The release of AMC fluorescent tag was measured using a cytofluorometer at 0 and 1 hour and specific activity was measured as the difference between 0 and 1 hour results and expressed as caspase activity per microgram protein. Each graph represents at least 5 separate experiments performed in duplicate (mean ± SEM). *P < .05 versus control at 0 hours; **P < .05 versus control at 24 hours. (A) Men, n = 2; women, n = 3. (B) Men, n = 3; women, n = 3. Eleanor J. Molloy et al. Blood 2003;102:2653-2659 ©2003 by American Society of Hematology

The release of cytochrome c from aging neutrophils on Western blot and densitometry. The release of cytochromecfrom aging neutrophils on Western blot and densitometry. Cytoplasmic fractions were extracted from human neutrophils after 0, 3, 6, 18, and 24 hours of aging in vitro. (A) Cytosolic cytochrome c release was assessed using Western blot analysis. The blot represents one of 3 separate experiments. (B) Densitometry of the 3 blots was performed and the density of each band (percent of total pixels) expressed as mean ± SEM. Eleanor J. Molloy et al. Blood 2003;102:2653-2659 ©2003 by American Society of Hematology

The effects of estradiol and progesterone on spontaneous cytochrome c release: Western blot and the expression of MnSOD in cytosolic and mitochondrial fractions. The effects of estradiol and progesterone on spontaneous cytochrome c release: Western blot and the expression of MnSOD in cytosolic and mitochondrial fractions. Cytoplasmic fractions were also extracted at 18 hours from control neutrophils and cells incubated with LPS, estradiol, progesterone, or both (10-8 g/mL). Cytosolic cytochrome c release was assessed using Western blot analysis. Cytoplasmic and mitochondrial fractions were also stained with MnSOD polyclonal antibody. Each blot represents 1 of 3 separate independent experiments. Eleanor J. Molloy et al. Blood 2003;102:2653-2659 ©2003 by American Society of Hematology