Inhibition of neutrophil apoptosis by LL-37. Neutrophils were incubated for 20 h over a range of LL-37 concentrations in the presence of 10% FBS. Modulation of spontaneous apoptosis was examined by FACS analysis and morphology. (A) Representative fields from cytospins. White arrows indicate examples of apoptotic neutrophils, 320× original magnification. (B–D) FACS analysis was used to determine the percentage of neutrophils, which were (B) apoptotic (FITC-annexin V-positive, propidium iodide-negative), (C) viable (FITC-annexin V-negative, propidium iodide-negative), and (D) necrotic (FITC-annexin V-positive, propidium iodide-positive). Figures represent the percentage of cells in LL-37-treated samples as a proportion of that observed in the vehicle alone-treated controls to correct for donor variation and indicate mean values ± sem, for n ≥ 6 for each condition, performed in duplicate, using seven different donors. Significance was assessed using absolute values in LL-37-treated samples compared with vehicle alone-treated controls by paired sample t-test analyses. *, P ≤ 0.05; **, P ≤ 0.01.

Modulation of neutrophil Mcl-1 expression and cleavage of BID in response to LL-37. Neutrophils were exposed to a concentration range of 0.25–50 μg/ml LL-37, 30 ng/ml GM-CSF as a positive control, or endotoxin-free water as a vehicle control for 4 h in the presence of 10% FBS. Whole cell protein lysates were prepared and analyzed by SDS-PAGE and Western immunoblotting. Immunoblots for expression of (A) Mcl-1 and (B) uncleaved BID are shown, each representative of n = 3 different donors, and expression of the housekeeping protein GAPDH was assessed as a loading control. Quantitative densitometry was performed, corrected for protein loading, expressed as a proportion of the vehicle alone-treated control sample, and displayed as mean values ± sem for n = 3 different donors. t-Test analyses were used to compare Mcl-1 or BID expression in LL-37- or GM-CSF-exposed samples with vehicle alone-treated controls. *, P ≤ 0.05.

LL-37-induced inhibition of neutrophil apoptosis involves multiple signaling pathways. Neutrophil apoptosis over 20 h incubation was examined in duplicate by FACS analysis for FITC-annexin V-positive, propidium iodide-negative cells after (A) incubation with 0.25 μg/ml or 1 μg/ml LL-37 or endotoxin-free water as a vehicle control, added 30 min after 100 μM-oxidized ATP, 10 μM WRW4, 200 ng/ml PTX, 50 μM PD098059, 5 μM wortmannin, or a vehicle-alone control in the presence of 10% FBS. Results represent the percentage of apoptotic cells as mean ± sem for n ≥ 4 per condition from five different donors. Paired sample t-test analyses were used to compare LL-37-treated samples with controls under the same inhibitor conditions. **, P ≤ 0.01, or (B) incubation with 1 μg/ml LL-37, 0.2 μM or 10 μM WKYMVm, or a vehicle-alone control. Results represent the percentage of apoptotic cells as mean ± sem for n = 3 from three different donors. Paired sample t-test analyses were used to compare treated samples with controls. **, P ≤ 0.01. (C) Neutrophil chemotaxis was assessed in triplicate in response to 10 μM WKYMVm or vehicle-alone control after preincubation with 10 μM WRW4 or vehicle-alone control, and the chemotactic index was displayed as mean ± sem for n = 3 from three different donors. Paired sample t-test analyses were used to compare WKYMVm-treated samples with controls and WRW4-pretreated samples with WKYMVm alone. *, P ≤ 0.05; **, P ≤ 0.01. (D) Neutrophil apoptosis over 20 h incubation was examined in duplicate by FACS analysis after incubation with 20 μg/ml GM-CSF or endotoxin-free water as a vehicle control, added 30 min after 10 μM or 50 μM PD098059 or vehicle-alone control. Results represent the percentage of apoptotic cells as mean ± sem for n = 3 per condition from three different donors. Paired sample t-test analyses were used to compare GM-CSF-treated samples with controls and PD098059-pretreated samples with GM-CSF alone. *, P ≤ 0.05; **, P ≤ 0.01.

Induction of caspase-dependent cell death in primary bronchial epithelial cells by LL-37. Primary human bronchial epithelial cells were exposed to (A) a concentration range of 1–100 μg/ml LL-37 or endotoxin-free water as a vehicle control, all in the presence of 10% FBS for 20 h or (B) pretreated with 50 μM caspase inhibitor I (Z-VAD-FMK) for 4 h prior to the addition of 25 μg/ml or 50 μg/ml LL-37 in the presence of 10% FBS for 20 h. Cells were fixed, and apoptosis was assessed by TUNEL assay. Three random fields of view, each containing more than 100 cells, were counted for each sample, and the percentage of TUNEL-positive cells in the field of view was expressed as a percentage of the number of DAPI-positive nuclei. Data represent means ± sem for n = 6 (A) or n = 3 (B). t-Test analyses were used to compare LL-37-treated samples with controls. *, P ≤ 0.05; **, P ≤ 0.01.

LL-37-mediated inhibition of neutrophil apoptosis occurs in the absence of cytolysis. Neutrophils were incubated over a range of LL-37 concentrations in the presence of 10% FBS for 0, 1, 4, or 20 h. (A–C) FACS analysis was used to determine the percentage of neutrophils that were apoptotic (FITC-annexin V-positive, propidium iodide-negative), viable (FITC-annexin V-negative, propidium iodide-negative), and necrotic (FITC-annexin V-positive, propidium iodide-positive) at (A) 1 h, (B) 4 h, and (C) 20 h. (D) Total cell counts were performed in duplicate using a haemocytometer and represented as a proportion of the number of cells in the vehicle-alone controls at each time-point. No significant difference was observed between control samples over the time course. Figures indicate mean values ± sem for n = 4 for each condition using four different donors. Significance was assessed using absolute values in LL-37-treated samples compared with vehicle alone-treated controls by paired sample t-test analyses. *, P ≤ 0.05; **, P ≤ 0.01.

Modulation of procaspase-3 cleavage in LL-37-treated neutrophils, which were exposed to a concentration range of 0.25–50 μg/ml LL-37, 30 ng/ml GM-CSF as a positive control, or endotoxin-free water as a vehicle control for 4 h in the presence of 10% FBS. Whole cell protein lysates were prepared and analyzed by SDS-PAGE and Western immunoblotting. (A) Immunoblots for expression of inactive procaspase-3 and active-cleaved caspase-3 are shown, representative of n = 5 different donors, and expression of the housekeeping protein GAPDH was assessed as a loading control. (B) Quantitative densitometry was performed, corrected for protein loading, expressed as a proportion of the vehicle alone-treated control sample, and displayed as mean values ± sem for n = 5 different donors. t-Test analyses were used to compare procaspase-3 and cleaved caspase-3 expression in LL-37- or GM-CSF-exposed samples with vehicle alone-treated controls. *, P ≤ 0.05; **, P ≤ 0.01.

Modulation of neutrophil cytokine responses by LL-37. The IL-8 or TNF-α production by neutrophils was assessed by ELISA analysis of culture supernatants following incubation of cells for 20 h in the presence of (A) 100 ng/ml LPS, 10 ng/ml IL-1β, 100 ng/ml TNF-α, or vehicle-alone control, with 10 μg/ml LL-37 or vehicle-alone control in the presence of 10% FBS; or (B) 100 ng/ml LPS, 50 ng/ml IL-1β, or vehicle-alone control with 10 μg/ml LL-37 or vehicle-alone control in the presence of 10% FBS. Data represent means ± sem for n ≥ 3 replicates per condition from four different donors. Paired sample t-test analyses were used to compare LL-37-treated samples with controls under the same stimulatory conditions. *, P ≤ 0.05; **, P ≤ 0.01.