The role of caspase activation and mitochondrial depolarisation in cultured human apoptotic eosinophils

Abstract

Caspases are key intracellular molecules in the control of apoptosis, but little is known concerning their relative contribution to the cascade of events leading to eosinophil apoptosis. We examined caspase-3, -8, and -9 activities in receptor ligation dependent apoptosis induction in the cultured eosinophils (CE). CE cultured alone for 48 hours exhibited constitutive apoptosis (12% ± 1.2). Significant (P < 0.05) enhancement of eosinophil apoptosis was observed following monoclonal antibody (Mab) treatment with CD45 (40% ± 0.7), CD95 (36% ± 1.6), or CD69 (34% ± 0.2). Caspase activity was analysed using the novel CaspaTagTM technique and flow cytometry. CE ligated with CD45 (Bra55), CD95 (Fas) and CD69 Mab resulted in caspase-3 and -9 activation after 16 hours post-ligation. This trend in caspase-3 and -9 activation continued to increase significantly through to the 20 and 24 hours time points when compared to isotype control. Activated up-stream caspase-8 was detected 16 and 20 hours after treatment with CD45, CD95 and CD69 Mab followed by a trend toward basal levels at 24 hours. Ligation of CD95 was followed by mitochondrial permeabilization, as demonstrated by marked increase in mitochondrial transmembrane potential ([Formula: see text]) at all time points. However, ligation with CD45 and CD69 failed to induce a change in [Formula: see text] at 16 hours post-treatment compared to isotype control even though there was an alteration in mitochondrial downstream-caspase activity following ligation with these Mab(s) at this time point. At 20 and 24 hours post-ligation, CD45 or CD69 induce significantly altered levels of [Formula: see text]. Thus, the intrinsic and extrinsic caspase pathways are involved in controlling receptor ligation-mediated apoptosis induction in human eosinophils, findings that may aid the development of a more targeted, anti inflammatory therapy for asthma.

Keywords: Apoptosis; Asthma; Caspases; Eosinophils; Mitochondria.

Figure 1

Flow cytometric analysis of apoptosis in cultured eosinophils. Representative experiment showing flow cytometric analysis of CE binding of annexin V-FITC (FL1-H) and uptake of PI (FL3-H) after incubation for 48 hours with dexamethasone (10⁻⁶ M) or Mab against pan-CD45 (BRA55), Fas (CD95), CD69, or an isotype control. Numbers in the upper right quadrant refer to annexin +/PI− (early apoptosis) and annexin +/PI+ (late apoptosis) CE.

Figure 2

Kinetics of changes in cultured eosinophil apoptosis. Time-course of effect of 10 μg/ml Mab-dependent ligation of pan-CD45 (Bra55), FAS (CD95), and CD69 or treatment with dexamethasone (10⁻⁶ M) on the constitutive rate of CE apoptosis compared with isotype control and untreated cells. Each point represents the mean ± SEM of four experiments (^∗P < 0.05 compared with isotype control or untreated cells in each case).

Figure 3

Caspase-3 activation in cultured eosinophils. Representative data from flow cytometry analysis of CE after incubation for 24 hours with Mab against pan-CD45, CD95, CD69, or an isotype control. The frequency histograms of number of events (y-axis) versus fluorescence intensity (x-axis) show two peaks. Caspase-3 negative (−) cells occur within the first log decade of the x-axis (FL1), whereas caspase-3 positive (+) cells are within the second and third log decade (M1).

Figure 4

Caspase activation in cultured eosinophils. Time-course of effect of Mab-dependent ligation of pan-CD45 (Bra55), FAS (CD95), and CD69 on CE caspase-3 (A), caspase-8 (B) or caspase-9 activity (C) compared with isotype control and untreated cells. Each bar represents the mean ± SEM of three separate experiments performed in duplicate (^∗P < .05 compared with isotype control).

Figure 5

$\mathrm{\Delta }{\mathrm{\Psi }}_m$ in cultured eosinophils undergoing apoptosis. (A) Representative example of bivariate JC-1 analysis of $\mathrm{\Delta }{\mathrm{\Psi }}_m$ in CE by flow cytometry. Distinct populations of cells with different extents of mitochondrial depolarisation are detectable in (A) isotype control and (B) following apoptosis inducing treatment with 10 μg/ml pan-CD45 (Bra55) Mab for 24 hours. (B) CE were ligated for varying time points with Mab against CD45, CD95, CD69, or an isotype control. Numbers indicate the percentage change in CE demonstrating a change in JC-1 binding characteristics (^∗P < .05 compared with isotype control, n = 4).