Journal:

Article Title: The Host Defense Peptide LL-37 Selectively Permeabilizes Apoptotic Leukocytes ▿

doi: 10.1128/AAC.01310-08

Figure Lengend Snippet: LL-37 can distinguish between membranes with different compositions. (A) The direct microbicidal activity of LL-37 (1.88, 3.75, and 7.5 μg/well) against E. coli was determined in an inhibition zone assay. LL-37 gave rise to clear, bacteria-free zones around the wells (upper panel), while sLL-37 (1.88, 3.75, and 7.5 μg/well) was inactive (lower panel). (B) Freshly separated neutrophils were not sensitive to the direct microbicidal, lytic action of LL-37 (100 μg/ml), as shown by the lack of 7-AAD fluorescence intensity plotted against the forward scatter. 7-AAD is a nucleic acid stain used for the detection of the loss of membrane integrity in cells. The results of one experiment with the percentage of cells in each region are shown. (C) LL-37 (5 ng/ml) readily permeabilized bacterial membrane-like liposomes (gray line) composed of PE-PG (7:3), while the eukaryotic membrane-like liposomes (black line), composed of PE-PC-cholesterol (1:1:1), were more resistant to the permeabilizing effect of LL-37. The percentage of total calcein released, as assessed by the addition of Triton X-100, is plotted against time. The means ± SDs (n = 3) obtained at 0, 50, 100, 150, and 200 s are shown. The amounts of calcein released at 100, 150, and 200 s were compared; and significant differences between the amounts released were was established (P < 0.01 at each point, as indicated [**]; n = 3). The arrow indicates the time of addition of peptide.

Article Snippet: Scrambled LL-37 (sLL-37; RSLEGTDRFPFVRLKNSRKLEFKDIKGIKREQFVKIL) was purchased from CSS-Albachem Ltd. (East Lothian, United Kingdom).

Techniques: Activity Assay, Inhibition, Fluorescence, Staining

Journal:

Article Title: The Host Defense Peptide LL-37 Selectively Permeabilizes Apoptotic Leukocytes ▿

doi: 10.1128/AAC.01310-08

Figure Lengend Snippet: Selective permeabilization of apoptotic neutrophils by LL-37. (A) Neutrophils incubated for 20 h spontaneously entered apoptosis, resulting in 66% viable cells and 31% apoptotic cells, as assessed by annexin V and 7-AAD staining and visualized by flow cytometry (left panel). When LL-37 (50 μg/ml) was added to these cells, all apoptotic (annexin V-positive) cells were permeabilized (7-AAD positive), while the viable (annexin V-negative) population was unaffected (middle panel) 5 min after addition of the peptide. sLL-37 (50 μg/ml) was inactive when it was added to the system (right panel). The results presented here represent those of one of three experiments performed for this experimental series. (B) Apoptosis was also assessed by CD16 shedding (left panel). Only the apoptotic neutrophils (CD16 negative) were permeabilized by the addition of LL-37 and stained positive for 7-AAD. The α-CD16 antibody also gained access to intracellular CD16 upon permeabilization, as shown by the increase in the fluorescence intensity of CD16 (right panel). The plots represent those from one of two independent experiments that were performed. Each plot shows the means ± SDs from one representative experiment.

Article Snippet: Scrambled LL-37 (sLL-37; RSLEGTDRFPFVRLKNSRKLEFKDIKGIKREQFVKIL) was purchased from CSS-Albachem Ltd. (East Lothian, United Kingdom).

Techniques: Incubation, Staining, Flow Cytometry, Fluorescence

Journal:

Article Title: The Host Defense Peptide LL-37 Selectively Permeabilizes Apoptotic Leukocytes ▿

doi: 10.1128/AAC.01310-08

Figure Lengend Snippet: Selective permeabilization of apoptotic NK cells by LL-37. (A) Isolated NK cells were incubated for 6 h and stained with annexin V and 7-AAD. Only a small amount of cells displayed apoptotic characteristics (4.7 ± 1.2%; n = 3). (B) When the cells were incubated in the presence of H2O2 (200 μM) for 6 h, the population of apoptotic NK cells increased (16.8 ± 3.3%; n = 3). The population of permeabilized NK cells also increased upon addition of H2O2 (from 5.3 ± 2.2% to 16.0 ± 4.2%; n = 3). (C) When LL-37 (5 μg/ml) was added to the H2O2-treated NK cells, the apoptotic population was permeabilized and stained positive for 7-AAD, in accordance with the results seen for neutrophils. At this concentration, no significant permeabilization of the viable population compared to that for the population treated with H2O2 alone was seen (P = 0.07; n = 3).

Article Snippet: Scrambled LL-37 (sLL-37; RSLEGTDRFPFVRLKNSRKLEFKDIKGIKREQFVKIL) was purchased from CSS-Albachem Ltd. (East Lothian, United Kingdom).

Techniques: Isolation, Incubation, Staining, Concentration Assay

Journal:

Article Title: The Host Defense Peptide LL-37 Selectively Permeabilizes Apoptotic Leukocytes ▿

doi: 10.1128/AAC.01310-08

Figure Lengend Snippet: The permeabilizing effect of LL-37 on neutrophils is concentration dependent and rapid. (A) Different concentrations of LL-37 ranging from 0 to 500 μg/ml were tested; increased concentrations led to an increased permeabilizing effect on the apoptotic cells, and at 500 μg/ml, a more general cytotoxic action that also affected viable neutrophils was observed. The percentage of cells is plotted against the LL-37 concentration; and the populations were viable (blue), apoptotic (red), and permeabilized (black). Mean ± SDs (n = 3) are shown. The results for each population were compared to those for an untreated sample, and significance was established, as indicated, by analysis of variance and Bonferroni's test for multiple comparisons. (B) Within 5 min after the addition of LL-37 (50 μg/ml), the permeabilizing action was complete. The membranes of viable cells retained their integrity for at least 5 min. The percentage of cells is plotted against time; and the populations were viable (blue), apoptotic (red), and permeabilized (black). Mean ± SDs (n = 3) are shown. Statistical significance was determined as indicated above for panel A. (C) After the gating of viable (green) and apoptotic (red) cells with the aid of annexin V staining (left panel), the increase in the intensity of 7-AAD staining after the addition of LL-37 (50 μg/ml) was monitored in real time. Only the apoptotic cells displayed an increase in 7-AAD intensity (right panel), and the permeabilization started immediately following addition of the peptide (arrow). The results represent those of one of two experiments that were performed. **, P < 0.01; ***, P < 0.001.

Article Snippet: Scrambled LL-37 (sLL-37; RSLEGTDRFPFVRLKNSRKLEFKDIKGIKREQFVKIL) was purchased from CSS-Albachem Ltd. (East Lothian, United Kingdom).

Techniques: Concentration Assay, Staining

Journal:

Article Title: The Host Defense Peptide LL-37 Selectively Permeabilizes Apoptotic Leukocytes ▿

doi: 10.1128/AAC.01310-08

Figure Lengend Snippet: The effect of LL-37 on neutrophils is independent of known receptors and Ca2+ signaling. (A) A mixed population of viable and apoptotic neutrophils was treated with the two antagonists WRW4 (5 μM) and oxATP (900 μM), which antagonize two known LL-37 neutrophil receptors, FPRL1 and P2X7, respectively. In the presence of the antagonists, LL-37 (50 μg/ml) still permeabilized apoptotic, annexin V-positive cells. Paired t tests were performed to establish statistical significance (n = 3). (B) An overnight preparation of incubated neutrophils was assessed for annexin V staining, and a mixed population of both viable (green) and apoptotic (red) neutrophils (left panel) was shown. The mixed population was stimulated with the potent neutrophil FPRL1 ligand WKYMVM (10−7 M), and the transient Ca2+ release was monitored by flow cytometry. The viable cells (green line) responded with a rise in the intracellular Ca2+ concentration, while the apoptotic cells (red line) were totally irresponsive. The results of one representative experiment are shown. The FL-1/FL-3 ratio was plotted against time (right panel). *, P < 0.05; ***, P < 0.001.

Article Snippet: Scrambled LL-37 (sLL-37; RSLEGTDRFPFVRLKNSRKLEFKDIKGIKREQFVKIL) was purchased from CSS-Albachem Ltd. (East Lothian, United Kingdom).

Techniques: Incubation, Staining, Flow Cytometry, Concentration Assay

Journal:

Article Title: The Host Defense Peptide LL-37 Selectively Permeabilizes Apoptotic Leukocytes ▿

doi: 10.1128/AAC.01310-08

Figure Lengend Snippet: The permeabilizing effect of LL-37 on apoptotic neutrophils is inhibited by human serum and HDL. (A) When HDL (650 μg/ml) was added to the inhibition zone plates, the clear zone surrounding the well after the addition of LL-37 (1.88 μg/well) decreased markedly. (B) In a control experiment, apoptotic neutrophils were readily permeabilized by the addition of LL-37 (50 μg/ml; left panel). When LL-37 was added to cells in a solution containing 10% normal human serum, the permeabilizing effect was totally blocked (middle panel). The addition of HDL (650 μg/ml) to the neutrophil population prior to the addition of LL-37 (50 μg/ml) also inhibited the permeabilizing effect of LL-37 on apoptotic neutrophils (right panel). Representative plots from three independent experiments and means ± SDs summarized from three independent experiments are shown.

Article Snippet: Scrambled LL-37 (sLL-37; RSLEGTDRFPFVRLKNSRKLEFKDIKGIKREQFVKIL) was purchased from CSS-Albachem Ltd. (East Lothian, United Kingdom).

Techniques: Inhibition

Journal:

Article Title: The Host Defense Peptide LL-37 Selectively Permeabilizes Apoptotic Leukocytes ▿

doi: 10.1128/AAC.01310-08

Figure Lengend Snippet: The selectivity of LL-37 for lysis of apoptotic cells is independent of PS exposure. Liposomes containing typical eukaryotic lipids were prepared without PS (green line) or with 10% PS (red line). LL-37 (5 ng/ml) was added, as indicated by the arrow, to the different liposomes, and calcein leakage was monitored over time. The introduction of PS in liposomes containing the phospholipids typically found in eukaryotic membranes had no effect. The percentage of total calcein released, as assessed by the addition of Triton X-100, is plotted against time. The ranges for the two data sets are shown. The total amount of calcein released over 225 s (determined from the area under the curve) was established and did not differ significantly (P = 0.08; n = 3) between the two types of liposomes.

Article Snippet: Scrambled LL-37 (sLL-37; RSLEGTDRFPFVRLKNSRKLEFKDIKGIKREQFVKIL) was purchased from CSS-Albachem Ltd. (East Lothian, United Kingdom).

Techniques: Lysis

Journal:

Article Title: The Host Defense Peptide LL-37 Selectively Permeabilizes Apoptotic Leukocytes ▿

doi: 10.1128/AAC.01310-08

Figure Lengend Snippet: Release of LDH and MPO from LL-37-treated apoptotic neutrophils. (A) Neutrophils were incubated together with or without the apoptosis inducer α-CD95; the result was 75% apoptotic cells and 45% apoptotic cells, respectively, in this set of experiments. These cells were treated with 50 μg/ml of LL-37 for 10 min, and the amount of the cytosolic enzyme LDH released was measured. It corresponded well with the fraction of apoptotic cells seen for each treatment, indicating the complete release of LDH from apoptotic neutrophils. Means + SDs (n = 3) are shown. Statistical significance was established by an unpaired t test. (B) The release of the intragranular matrix protein MPO was also measured in α-CD95-treated neutrophils. The amount of MPO released was significantly higher for LL-37-treated cells than for untreated cells. Means + SDs (n = 3) are shown. A paired t test was used for statistical analysis. **, P < 0.01.

Article Snippet: Scrambled LL-37 (sLL-37; RSLEGTDRFPFVRLKNSRKLEFKDIKGIKREQFVKIL) was purchased from CSS-Albachem Ltd. (East Lothian, United Kingdom).

Techniques: Incubation