Mechanisms of cell death induced by the neutrophil antimicrobial peptides alpha-defensins and LL-37.
ABSTRACT The aim of this study was to investigate the mechanisms of cell death mediated by the antimicrobial peptides neutrophil defensins (human neutrophil peptides 1-3 [HNP1-3]) and LL-37.
HNP1-3- and LL-37-mediated cell death was assessed in human lung epithelial cells and Jurkat T-cells in serum-free culture media.
Both HNP1-3 and LL-37 induced cell death in Jurkat T-cells and A549 cells. HNP1-3 but not LL-37 induced caspase-3/-7 activity and caused cleavage of [ADP-ribose] polymerase (PARP) in Jurkat cells, while in A549 cells neither peptides induced caspase-3/-7 activation. Furthermore, both peptides increased mitochondrial cytochrome c release in A549 and Jurkat cells. Our observation that over-expression of the anti-apoptotic protein Bcl-2 in Jurkat cells did not affect HNP1-3- or LL-37-induced cell death indicates that antimicrobial peptide-induced cytochrome c release is not involved in peptide-induced cell death. Finally, in A549 cells and in primary bronchial epithelial cells, both HNP1-3 and LL-37 induced DNA breaks as demonstrated by increased TUNEL labelling.
The results from this study suggest that the antimicrobial peptides HNP1-3 and LL-37 induce cell death, which is associated with mitochondrial injury and mediated via different intracellular pathways.
- SourceAvailable from: erj.ersjournals.com[show abstract] [hide abstract]
ABSTRACT: The human lung is exposed to a large number of airborne pathogens as a result of the daily inhalation of 10,000 litres of air. The observation that respiratory infections are nevertheless rare is testimony to the presence of an efficient host defence system at the mucosal surface of the lung. The airway epithelium is strategically positioned at the interface with the environment, and thus plays a key role in this host defence system. Recognition systems employed by airway epithelial cells to respond to microbial exposure include the action of the toll-like receptors. The airway epithelium responds to such exposure by increasing its production of mediators such as cytokines, chemokines and antimicrobial peptides. Recent findings indicate the importance of these peptides as effector molecules of innate immunity by killing microorganisms, but also as regulators of inflammation, immunity and wound repair. Finally, the clinical relevance of the functions of the airway epithelium in innate immunity is discussed.European Respiratory Journal 03/2004; 23(2):327-33. · 6.36 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: We identified antibacterial components in human T and natural killer (NK) cells by using freshly isolated lymphocytes enriched for T and NK cells as starting material. After growing these lymphocytes for 5 days in the presence of interleukin (IL)-2, we isolated and characterized several antibacterial peptides/proteins from the supernatant-alpha-defensins (HNP 1-3), LL-37, lysozyme, and a fragment of histone H2B-although other active components were also present. We then used reverse transcriptase-polymerase chain reaction to search for expression of the gene coding for LL-37 in several B-cell lines, gammadelta T-cell lines, NK clones, and one monocytic cell line, with positive results, but found no expression in several alphabeta T-cell lines. The alpha-defensins (HNP 1-3) were also found to be expressed in several of these cell lines. To confirm the presence of these antibacterial peptides in lymphocytes, we localized them to NK, gammadelta T cells, B cells, and monocytes/macrophages by using double-staining immunohistochemical analysis of freshly isolated lymphocytes. We also found that primary cultures of lymphocytes transcribe and secrete LL-37 and that these processes are affected by IL-6 and interferon-gamma. In addition, we demonstrated that LL-37 has chemotactic activity for polymorphonuclear leukocytes and CD4 T lymphocytes, whereas others have shown chemotactic activity for human alpha-defensins (HNP 1-2). These findings suggest that microbicidal peptides are effector molecules of lymphocytes and that antibacterial activity previously shown to be derived from T and NK cells may be partly mediated by the antibacterial peptides LL-37 and HNP 1-3.Blood 12/2000; 96(9):3086-93. · 9.06 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Cytotoxic lymphocytes, NK cells and CD8(+) T cells play a pivotal role in the host defense. To reveal the biological function of these cells through establishing a comprehensive gene expression profile, serial analysis of gene expression was performed in human peripheral blood NK cells and CD8(+) T cells. In total, 85,848 tags corresponding to >20,000 different transcripts were sequenced. The genes expressed abundantly in these libraries mostly consisted of genes encoding MHC class I and molecules related to protein synthesis. Among gene transcripts which related to cytotoxicity, granulysin, perforin, granzyme B and alpha-defensin 1 were highly expressed in NK cells. Resting CD8(+) T cells did not express the genes related to cytotoxicity, but expressed abundantly the genes encoding chemokines, tumor necrosis factor family. When CD8(+) T cells were sorted into naive, memory and effector subsets based on the expression of CD45RA and CD27, perforin and granzyme B were expressed in the CD45RA(+)CD27(-) effector subset. Alpha-defensin 1, one of the selectively expressed genes in NK cells, induced migration of naive CD8(+)CD45RA(+)CD27(+) T cells, but not memory CD8(+)CD45RA(-)CD27(+) or effector CD8(+)CD45RA(+)CD27(-) T cells. Furthermore, treatment with IL-15, a stimulator of NK cell development, differentiation, survival and cytotoxicity, rapidly enhanced the expression of alpha-defensin 1 in NK cells. The identification of the genes preferentially expressed in NK and CD8(+) T cell subsets may give important insights into the functions of these cells against virus infection and in tumor immunity.International Immunology 11/2002; 14(10):1085-98. · 3.14 Impact Factor
Infl amm. res. 55 (2006) 119–127
Infl ammation Research
© Birkhäuser Verlag, Basel, 2006
Mechanisms of cell death induced by the neutrophil
antimicrobial peptides a-defensins and LL-37
J. Aarbiou1,†, G. S. Tjabringa1,†, R. M. Verhoosel1, D. K. Ninaber1, S. R. White3, L. T. C. Peltenburg2, +, K. F. Rabe1 and
P. S. Hiemstra1
1 Department of Pulmonology, C3-P, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands, Fax: ++31 71 5266927,
2 Clinical Oncology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands
3 Section of Pulmonary and Critical Care Medicine, Department of Medicine, Division of Biological Sciences, The University of Chicago, Chicago,
Received 8 July 2005; returned for revision 21 October 2005; returned for fi nal revision 8 December 2005;
accepted by M. Parnham 14 December 2005
Abstract. Objective: The aim of this study was to investigate
the mechanisms of cell death mediated by the antimicrobial
peptides neutrophil defensins (human neutrophil peptides 1-
3 [HNP1-3]) and LL-37.
Materials and methods: HNP1-3- and LL-37-mediated cell
death was assessed in human lung epithelial cells and Jurkat
T-cells in serum-free culture media.
Results: Both HNP1-3 and LL-37 induced cell death in Ju-
rkat T-cells and A549 cells. HNP1-3 but not LL-37 induced
caspase-3/-7 activity and caused cleavage of [ADP-ribose]
polymerase (PARP) in Jurkat cells, while in A549 cells nei-
ther peptides induced caspase-3/-7 activation. Furthermore,
both peptides increased mitochondrial cytochrome c release
in A549 and Jurkat cells. Our observation that over-expres-
sion of the anti-apoptotic protein Bcl-2 in Jurkat cells did not
affect HNP1-3- or LL-37-induced cell death indicates that
antimicrobial peptide-induced cytochrome c release is not
involved in peptide-induced cell death. Finally, in A549 cells
and in primary bronchial epithelial cells, both HNP1-3 and
LL-37 induced DNA breaks as demonstrated by increased
Conclusions: The results from this study suggest that the
antimicrobial peptides HNP1-3 and LL-37 induce cell death,
which is associated with mitochondrial injury and mediated
via different intracellular pathways.
Key words: Antimicrobial peptides – Defensins – Cathelici-
dins – Cytotoxicity – Caspases
Neutrophil-derived antimicrobial peptides like neutrophil a-
defensins and LL-37 play a central role in innate immunity. In
addition to killing invading microbes, these peptides have sev-
eral other functions, including chemotactic activity, activation
of various cell types and cytotoxic activity towards eukaryotic
cells . Limited studies demonstrate pathways involved in
cytotoxic activities of these antimicrobial peptides.
Neutrophil defensins are small cationic polypeptides
of the a-defensin subfamily that are expressed mainly in
neutrophils but also in specifi c subsets of T-cells, monocytes
and NK-cells [2–4]. Four neutrophil defensins have been
identifi ed (human neutrophil peptides 1-4 [HNP1-4]) that
are stored in large amounts in azurophilic granules and are
released upon neutrophil activation . Neutrophil defensin
levels of up to 1.6 mg/ml have been described in airway
secretions from patients with infl ammatory lung diseases,
such as cystic fi brosis [5, 6]. The cationic peptide LL-37 is
the single human member of the cathelicidin family of anti-
microbial peptides, which was fi rst identifi ed in neutrophils
, and later also shown to be expressed in various epithelia
[8–10], lymphocytes , monocytes  and mast cells .
LL-37 is produced in a precursor form, designated human
cathelicidin antimicrobial protein 18 (hCAP-18) that is pro-
teolytically cleaved by proteinase 3 from human neutrophils
, and by gastricsin present in human seminal fl uid .
In addition to antimicrobial activity, both neutrophil de-
fensins and LL-37 were demonstrated to display a variety
of functions involved in infl ammatory and immunological
processes [5, 14, 15].
At high concentrations, both neutrophil defensins and
LL-37 have been shown to display cytotoxic activities against
eukaryotic cells, including airway epithelium [16–18]. In ad-
dition, HNP1-3 was demonstrated to induce DNA breaks in
leukemia cell lines . Membrane disturbance was demon-
Correspondence to: P. S. Hiemstra
† J. Aarbiou and G.S. Tjabringa contributed equally to this manuscript
+ Present address: Crucell Holland B.V., Leiden, The Netherlands
120 J. Aarbiou et al. Infl amm. res.
Effects of HNP1-3 and LL-37 on the viability of epithelial cells was
assessed by measuring mitochondrial activity using (4.5-dimethylthi-
azol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assays as previously
described . Briefl y, A549 cells were cultured to near confl uence in
96-well tissue culture plates and subsequently serum-deprived over-
night. After stimulation for 24 h with various concentrations of HNP1-3
or LL-37 in serum-free medium, the cells were incubated with 5 mg/ml
MTT in PBS for 2 h, lysed in extraction buffer (20% (w/v) SDS, 50 %
(v/v) N,N-dimethyl formamide, 2 % (v/v) acetic acid, pH 4.7) overnight
and optical density at 562 nm was measured.
FACS analysis PI/Annexin V
Cell death in Jurkat cells was measured by FACS analysis using propid-
ium iodide (PI) exclusion . Jurkat cells were incubated in serum-
free medium for 30 minutes and subsequently incubated for 16 h with
serum-free medium alone or supplemented with HNP1-3 or LL-37 at
concentrations as indicated. As a positive control, the chemotherapeutic
agent etoposide (Sigma Aldrich) was used. Etoposide is an inhibitor
of DNA topoisomerase II, and causes single and double strand breaks
in cellular DNA, resulting in apoptosis. The effect of the pan-caspase
inhibitor benzyloxycarbonyl-Val-Ala-Asp-fl uoromethyl ketone (zVAD;
Bachem, Bubendorf, Switzerland) was analyzed by preincubating cells
with 50 µM zVAD for 1 h prior to addition of etoposide, HNP1-3 or LL-
37. After washing in PBS, the cells were incubated with 1 µg/ml PI (Mo-
lecular Probes, Leiden, The Netherlands) in 0.5 % (w/v) bovine serum
albumin in PBS and incubated for 10 min in the dark on ice. To exclude
cellular fragments, viable and dead cells were gated and analyzed on a
FACS sorter (BD Biosciences, San Jose, CA).
Apoptosis in Jurkat T-cells by HNP1-3 and LL-37 was determined
by FACS analysis using FITC-labeled Annexin V (Nexin Research,
Hoeven, The Netherlands) and PI according to established methods
. Jurkat cells were incubated in serum-free medium for 30 min and
subsequently incubated for 3 and 16 h with serum-free medium alone
or supplemented with etoposide (Sigma Aldrich), HNP1-3, or LL-37 at
concentrations as indicated. After incubation with FITC-labeled annexin
V and PI, analysis was performed on a FACS sorter (BD Biosciences).
Caspase-3/-7 activity assay
Caspase-3/-7 activity in Jurkat and A549 cell lysates was measured us-
ing the Ac-DEVD-AFC (7-amino-4-trifl uoromethyl-coumarin) peptide
substrate (Alexis, Nottingham, UK). For these experiments, cells were
plated on 6-well tissue culture plates and cultured to near confl uence
as described. After serum-deprivation for 30 min, cells were stimulated
with serum-free medium alone or supplemented with etoposide (50 µM),
HNP1-3 (50 µg/ml) or LL-37 (60 µg/ml) for the indicated time periods.
Next, the cells were washed and lysed in lysis buffer (1 % [w/v] 3-[(3-
150 mM NaCl, 10 mM HEPES, 1 mM phenylmethylsulfonyl fl uoride
(PMSF) and 1 mM Na3VO4; pH 7.4), and cellular lysates were cleared
by centrifugation. Ac-DEVD-AFC was dissolved in dimethylsulfoxide
and further diluted to 100 µM in 20 mM PIPES, 100 mM NaCl, 1 mM
EDTA, 0.1 % (w/v) CHAPS, 10 % (w/v) sucrose and 10 mM dithiotreitol
(Caspase buffer, pH 7.2). Eighty µl of the AC-DEVD-AFC solution was
added to 10 µg cell lysate (in 20 µl lysis buffer). Caspase-3/-7 activity
was determined by measuring AFC-fl uorescence (excitation at 400 nm;
fl uorescence emission at 505 nm) during 60 min at 30 °C, using the Vic-
tor 2 multilabel counter 1420 (Wallac, Turku, Finland).
Analysis of PARP cleavage
For the detection of PARP cleavage, cells were stimulated and lysed
as described for caspase-3/-7 activation assays. Fifteen µg total protein
strated to be mediated via pore formation for both neutrophil
defensins and LL-37 [20, 21]. However, while neutrophil
defensins were suggested to enter the target cell, LL-37 was
suggested to stick into the membrane [20, 22–24]. Although
interactions of neutrophil defensins and LL-37 with the cellu-
lar membrane have been described, the intracellular pathways
involved in cell death induction remain largely unknown.
The aim of this study was to investigate the mechanisms
involved in cell death induction by neutrophil defensins
and LL-37. Jurkat cells are widely used to study cell death.
Cytotoxic pathways in these cells are readily induced by a
wide range of stimuli and have been extensively character-
ized. Therefore, effects of neutrophil defensins and LL-37
on activation of caspase-3/-7 and cleavage of their substrate
poly [ADP-ribose] polymerase (PARP), mitochondrial cy-
tochrome c release and on induction of DNA breaks were
studied in both Jurkat cells and lung epithelial cells.
Materials and methods
Human neutrophil defensins were isolated from neutrophil granules as a
mixture of HNP-1, -2 and -3 as previously described . LL-37 (amino
acid sequence LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES)
was synthesized by solid phase peptide synthesis on a TentagelS AC
(Rapp, Tuebingen, Germany) using 9-fl uorenylmethoxycarbonyl
(Fmoc)/tBu chemistry, benzotriazole-1-yl-oxy-tris-pyrrolidino-phospho-
nium hexafl uorophosphate (PyBOP)/ N-methylmorpholine (NMM) for
activation and 20 % piperidine in N-methylpyrrolidone (NMP) for Fmoc
removal. The peptide was cleaved from the resin and deprotected with
trifl uoroacetic acid (TFA)/ water and purifi ed by reverse-phase high per-
formance liquid chromatography (RP-HPLC) on a Vydac C18 column.
The molecular mass was confi rmed by Maldi-Tof mass spectrometry.
Cells from the A549 human lung carcinoma cell line and Jurkat T-cells
were obtained from the American Type Culture Collection (ATCC,
Manassas, VA). Bcl-2- and empty vector-transfected Jurkat T-cells
(clone J16)  were kindly provided by J. Borst (Netherlands Cancer
Institute, Amsterdam, The Netherlands). The cells were routinely cul-
tured in RPMI 1640 medium (Gibco, Grand Island, NY), supplemented
with 2 mM L-glutamine, 20 U/ml penicillin, 20 µg/ml streptomycin (all
BioWhittaker, Walkersville, MD) and 10 % (v/v) heat-inactivated FCS
(Gibco) at 37 °C in a 5 % CO2-humidifi ed atmosphere.
Subcultures of primary bronchial epithelial cells (PBEC) were
obtained from resected lung tissue from patients that underwent a
pneumectomy or lobectomy for lung cancer at the Leiden University
Medical Center. Bronchial epithelial cells were isolated essentially as
previously described  using enzymatic digestion with 0.1 % (w/v)
proteinase XIV (Sigma Aldrich, St. Louis, MO). The cells were cul-
tured under submerged conditions in keratinocyte serum-free medium
(KSFM, Gibco), supplemented with 0.2 ng/ml epidermal growth factor
(EGF, Gibco), 25 µg/ml bovine pituitary extract (BPE, Gibco), 1 µM
isoproterenol, 20 U/ml penicillin, 20 µg/ml streptomycin at 37 °C in a
5 % CO2-humidifi ed atmosphere. PBEC were cultured in tissue cul-
ture plates precoated with 10 µg/ml fi bronectin (isolated from human
plasma), 30 µg/ml Vitrogen (Cohesion technologies Inc., Palo Alto,
CA) and 10 µg/ml bovine serum albumin (Sigma Aldrich). When cells
reached near confl uence, they were incubated in KSFM medium without
isoproterenol in the presence of 1 mM Ca2+ and 5 nM all-trans retinoic
acid (Sigma Aldrich) for 36 h before stimulation of the cells for various
Vol. 55, 2006 Neutrophil antimicrobial peptides and cell death 121
of the lysates was separated on 10 % SDS-PAGE gels (Mini-protean 3
system, Biorad, Hercules, CA) and transferred to polyvinylidene difl uo-
ride membranes using the Mini-transblot system (Biorad). Protein levels
in the test samples were quantifi ed by bicinchonic acid (BCA) protein
reagent detection (Pierce, Rockford, IL), and equal protein amounts
were loaded on the SDS-PAGE gels. Non-specifi c binding sites were
blocked, and the membranes were incubated with rabbit-anti-PARP
polyclonal antibodies (New England Biolabs, Beverly, MA) and horse-
radish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies
(Jackson, West Grove, PA). Immunoreactivity was detected using the
electrochemiluminescent (ECL) Western blotting detection reagents
(Amersham Pharmacia Biotech, Uppsala, Sweden).
Cytochrome c release
Cytoplasmic cytochrome c was determined using Western blot analy-
sis. After serum deprivation for 30 min, Jurkat and A549 cells were
incubated with serum-free medium alone or supplemented with 50 µM
etoposide, 50 µg/ml HNP1-3 or 60 µg/ml LL-37 for the indicated time
periods. Stimulated cells were then collected, washed in PBS, resus-
pended in ice-cold cell extraction buffer (10 mM HEPES pH 7.4, 50 mM
KCl, 5 mM EGTA, 5 mM MgCl2, 300 mM sucrose, 1 mM dithiotreitol,
100 µM cytochalasin B, 1 mM PMSF) and incubated on ice for 30 min
to allow swelling. Swollen cells were lysed by resuspension through a
26G1/2 needle (BD Biosciences). After removal of nuclei and intact
cells by centrifugation in a microfuge for 5 min at 2,000 rpm, the heavy
membrane and cytoplasmic fractions were separated by centrifugation
for 5 min at 13,000 rpm. Protein concentrations in the cytoplasmic frac-
tions were determined using the Bradford method (Bio-Rad,), and 2 µg
total protein was separated on 15 % SDS-PAGE gels. Western blot pro-
cedures were continued as described for PARP cleavage detection, using
mouse monoclonal antibodies against cytochrome c (BD Pharmingen,
Franklin Lake, NJ) and HRP-conjugated anti-mouse secondary antibod-
ies (DAKO, Glostrup, Denmark).
For the detection of TUNEL-positive cells, PBEC were cultured on
precoated 4 chamber tissue culture glass slides (BD Labware, Fran-
klin Lake, NJ). After growth factor deprivation for 30 min in KSFM
medium containing 1 mM Ca2+ and 5 nM retinoic acid, the cells were
stimulated in the same medium. A549 cells were incubated for 30 min
in serum-free medium before stimulation. After incubation of PBEC or
A549 cells with medium alone or supplemented with 50 µg/ml HNP1-3,
30 µg/ml LL-37 for 5 or 24 h, the cells were washed and subsequently
fi xed overnight in 10 % (v/v) formalin. Incubation of cells with 0.5 µg/ml
cytochalasin-D was used as a positive control .
DNA breaks in fi xed monolayers were demonstrated by labelling
free 3'-hydroxyl groups of DNA using a Trevigen TUNEL fl uorescent
assay kit. Slides were counterstained in 1 mM Hoechst 33258 for 45 s and
visualized immediately by fl uorescent microscopy. Representative imag-
es were collected using a Sensys 12-bit cooled CCD camera (Photomet-
rics, Inc., Tucson, AZ) connected to a Nikon fl uorescence microscope.
Fields were selected at random by one investigator who was different
from the investigator performing the experiment. TUNEL-positive nuclei
and Hoechst-stained nuclei were counted in each image as the area of the
nuclei in pixels after setting a threshold to exclude extraneous positive
pixels using Spectrum IP software (IP Labs, Vienna, VA) on a Macintosh
computer. TUNEL-positive cells were expressed as the percentage of the
thresholded area of the TUNEL-stained image divided by the thresholded
area of the Hoechst-stained image, as previously described .
Results are expressed as mean ±SEM. Data obtained from at least
three separate experiments were analyzed for statistical difference by
the Student’s t-test for paired samples and differences were considered
signifi cant when P < 0.05.
Effects of HNP1-3 and LL-37 on death of non-adherent Ju-
rkat cells were measured by FACS analysis using PI exclu-
sion assays (Fig. 1A). Increased numbers of PI-positive cells
were observed with HNP1-3 at 25 µg/ml and with LL-37 at
10 µg/ml. Incubation with higher concentration of HNP1-3
and LL-37 resulted in even higher numbers of PI-positive
cells. To study apoptosis, the effects of HNP1-3 and LL-37
on both Annexin V and PI staining in Jurkat cells were de-
termined by FACS analysis. Cells undergoing programmed
cell death may translocate phosphatidylserine from their
inner cytoplasmic membrane to their cell surface, Annexin
V binds to this phosphotidylserine. While HNP1-3 was dem-
onstrated to increase PI/Annexin V double positive cells,
which are suggested to be late apoptotic or necrotic cells,
LL-37 increased PI positive cells without affecting Annexin
V (Fig. 1B). Since LL-37, like most antimicrobial peptides,
is a cationic peptide that binds well to cellular membranes,
it is possible that LL-37 blocks annexin V binding. This may
explain the unexpected results observed with LL-37 in the
PI/annexin V experiments. Etoposide, which was used as
a positive control, increased PI/Annexin V double positive
Cytotoxic effects of HNP1-3 and LL-37 on adher-
ent A549 cells were determined by measuring metabolic
activity using the tetrazolium salt MTT assay (Fig. 2).
Incubation of cells with HNP1-3 for 24 h at concentrations
of 50 µg/ml and higher resulted in decreased metabolic
activity, while LL-37 decreased mitochondrial activity at
concentrations of 25 µg/ml and higher. These results con-
fi rm the previously observed cytotoxic effects of HNP1-3
and LL-37 [16–18].
Apoptotic cell death is characterized by the activation of
caspases, including the effector caspases caspase-3/-7. Gen-
eration of caspase-3/-7 activity in cellular lysates due to the
action of HNP1-3 and LL-37 was determined by measuring
fl uorescence of the AFC fl uorochrome after cleavage from
the caspase-3/-7 substrate peptide DEVD. Incubation of
Jurkat cells with HNP1-3 or etoposide for various time pe-
riods resulted in increased caspase-3/-7 activity after 16 and
24 h, while incubation with medium alone or with LL-37
did not (Fig. 3A). No signifi cant caspase-3/-7 activity was
detected in cellular lysates prepared from A549 cells that
were incubated with HNP1-3 or LL-37 (data not shown).
Also etoposide did not induce apoptosis in A549 cells,
which may indicate that these cells are less susceptible to
apoptotic pathways than Jurkat T-cells. PARP, a polymer-
ase involved in DNA repair, is cleaved following caspase-3
mediated cleavage. To study the effect of HNP1-3 and LL-
37 on cleavage of PARP, Jurkat cells were incubated with
122 J. Aarbiou et al. Infl amm. res.
etoposide, HNP1-3 or LL-37 for 16 h, after which PARP
cleavage was determined using Western blot analysis (Fig.
3B). Both HNP1-3 and etoposide, which were demonstrated
to induce caspase activity (Fig. 3A), cleaved PARP in Jurkat
cells. In agreement with the lack of caspase activity after
treatment with LL-37, also no PARP cleavage was observed
in these cells.
Effect of ZVAD on HNP1-3- and LL-37 induced cell
To determine whether caspase activity is involved in the
observed HNP1-3-induced cell death, Jurkat cells were pre-
incubated with the caspase inhibitor zVAD. As shown by PI
exclusion assays, HNP1-3 and LL-37 showed a high percent-
age of cell death that was not inhibited by zVAD, (Fig. 4A).
Etoposide-induced cell death was marginally inhibited by
zVAD. As a control, zVAD completely blocked HNP1-3 and
etoposide-induced caspase-3/-7 activity, as well as baseline
activity (Fig. 4B).
medium2550 100 1025 100
% PI positive cells
Fig. 1. Effects of HNP1-3 and
LL-37 on Jurkat cell viability.
Jurkat T-cells were incubated for
30 min in serum-free medium,
and stimulated with various con-
centrations of HNP1-3 or LL-37
in serum-free medium. (A) Cell
death was measured by PI ex-
clusion assays after incubation
for 16 h with medium alone or
supplemented with HNP1-3 or
LL-37 at concentrations as indi-
cated. Data are mean ± SEM of 3
separate experiments performed
in triplicate. *p < 0.05 versus
medium alone. (B) Effects of
HNP1-3 and LL-37 on Annexin
V and PI staining in Jurkat cells.
Jurkat cells were incubated with
HNP1-3 (50 µg/ml) or LL-37
(60 µg/ml) for 3 and 16 h, and
FACS analysis was performed
using PI and FITC-labeled An-
25 50100 1025 100
Fig. 2. Effect of HNP1-3 and LL-37 on A549 cell mitochondrial activ-
ity. A549 cells were cultured overnight in serum-free medium and stimu-
lated the next day with various concentrations of HNP1-3 and LL-37 in
serum-free medium. After 24 h, mitochondrial activity was measured by
MTT colorimetric OD562 measurement.
Vol. 55, 2006 Neutrophil antimicrobial peptides and cell death 123
Cytochrome c release
Mitochondria have been demonstrated to play a central role
in cell death induction. To study their involvement in HNP1-
3- and LL-37-induced cell death, release of cytochrome c
from the mitochondria into the cytosol after incubation with
HNP1-3 or LL-37 was determined in both Jurkat cells and
A549 cells (Fig. 5). Incubation of Jurkat cells with serum-
free medium resulted in a moderate increase of cytoplasmic
cytochrome c content after 24 h, whereas the pro-apoptotic
agent etoposide showed a marked increase after 16 h, which
further increased until 24 h. Incubation of Jurkat cells with
HNP1-3 caused an increase in the cytoplasmic cytochrome
c content already after 3 h, which persisted for 16 h. In con-
trast, LL-37 showed a slight increase only after 3 h. In A549
cells both HNP1-3 and LL-37 increased cytoplasmic cyto-
chrome c protein after 3 and 6 h of incubation. In contrast
to Jurkat cells, incubation of A549 cells with medium alone
did not result in cytochrome c release, while incubation with
etoposide resulted in a slight increase.
Relative DEVD activity
Fig. 3. (A) Effect of HNP1-3 and LL-37 on caspase-3/-7 activation in
Jurkat cells. Cells were incubated with serum-free medium for 30 min,
and stimulated with serum-free medium alone (closed squares), or sup-
plemented with 50 µM etoposide (closed diamonds), 50 µg/ml HNP1-3
(open circles) or 60 µg/ml LL-37 (open triangles) for various time pe-
riods, and caspase-3/-7 activity in cellular substrates was determined
using the AC-DEVD-AFC peptide substrate. (B) Effects of HNP1-3
and LL-37 on PARP-cleavage in Jurkat cells. Cells were incubated for
30 min in serum-free medium, and stimulated for 16 h with serum-free
medium alone, or supplemented with etoposide (etop; 50 µM), HNP1-3
(50 µg/ml) or LL-37 (60 µg/ml), and PARP cleavage in cellular lysates
was determined by Western blot analysis. Data in A and B are repre-
sentatives of three individual experiments.
% PI positive cells
Relative DEVD activity
Fig. 4. (A) Effect of zVAD on HNP1-3- and LL-37-induced cell death in
Jurkat cells measured by PI exclusion assays. Jurkat cells were incubat-
ed for 30 min with serum-free medium, and stimulated with serum-free
medium alone, or supplemented with etoposide (etop; 50 µM), HNP1-3
(50 µg/ml) or LL-37 (60 µg/ml) following incubation with (+; solid bars)
or without (-; open bars) the caspase-inhibitor zVAD (50 µM). Data are
mean ±SEM of 4 independent experiments performed in triplicate. (B)
Effect of zVAD on caspase-3/-7 activity. Jurkat cells were incubated for
30 min in serum-free medium, and stimulated with serum-free medium
alone, or supplemented etoposide, HNP1-3 or LL-37 following incuba-
tion with or without zVAD as in A, and caspase-3/-7 activity in cellular
substrates was determined using the AC-DEVD-AFC peptide substrate.
The experiment was performed in triplicate, and similar results were
obtained in another experiment.
124 J. Aarbiou et al. Infl amm. res.
Members of the Bcl-2 family have been demonstrated
to regulate cell death. While members such as Bax and Bak
may induce cell death by forming pores in the mitochondrial
membrane, Bcl-2 blocks this cytochrome c release and there-
by is thought to inhibit the induction of apoptosis. To study
whether this protein family is involved in HNP1-3- and LL-
37-induced cell death, Jurkat cells stably transfected with
Bcl-2 were incubated with etoposide, HNP1-3 or LL-37.
HNP1-3- and LL-37-induced cell death in Bcl-2-transfected
cells was comparable to death of cells transfected with the
empty vector (Fig. 6), suggesting that HNP1-3- and LL-37-
induced cell death is not mediated by Bcl-2. As expected,
cell death induced by etoposide treatment was lower in Bcl-
To determine if HNP1-3 and LL-37-induced cell death was
accompanied by DNA fragmentation, the effect of HNP1-3
and LL-37 on induction of DNA breaks was determined by
DNA nick-end labeling. Upon incubation of A549 cells (Fig.
2.2 3.0 1.3 2.2
3.1 2.1 1.3 1.2
1.3 1.4 1.4 2.0
1 1.0 1.1 1.2
1.7 1.1 1.0 1.6
3.1 2.6 2.5 1.6
1.4 0.9 2.0 3.1
1 0.9 1.3 2.1
Time (h) 3 6 16 24
Time (h) 3 6 16 24
Fig. 5. Effects of HNP1-3 and LL-37 on cytochrome c release in Jurkat
cells and A549 cells. Cells were incubated for 30 min in serum-free me-
dium, and stimulated for various time periods with serum-free medium
alone or supplemented with etoposide (etop; 50 µM), HNP1-3 (50 µg/ml)
or LL-37 (60 µg/ml) and cytochrome c content in the cytosolic fraction
was determined by Western blot analysis. Values indicate fold-increase
as compared to serum-free medium-treated cells for 3 h obtained by
quantitative densitometry. Data are representative of three individual
% PI positive cells
Fig. 6. HNP1-3- and LL-37-induced cell death in Jurkat cells overex-
pressing Bcl-2, measured by PI exclusion assays. Jurkat cells stably
transfected with an empty vector (wt; open bars) or with Bcl-2 (Bcl;
solid bars) were incubated for 30 min in serum-free medium, and
stimulated for 16 h with serum-free medium alone or supplemented with
etoposide (50 µM), HNP1-3 (50 µg/ml) or LL-37 (60 µg/ml). Data are
mean ±SEM of 3 independent experiments performed in triplicate. ns:
not signifi cant.
medium HNP1-3LL-37 cyt-D
% TUNEL positive cells
% TUNEL positive cells
Fig. 7. Effects of HNP1-3 and LL-37 on DNA fragmentation in A549
cells (A) and PBEC (B) as determined by TUNEL assay. Cells were
incubated with medium alone or supplemented with HNP1-3 (50 µg/ml),
LL-37 (30 µg/ml) or cytochalasin D (cyt-D; 0.5 µg/ml) for 5 h (open
bars) or 24 h (solid bars), and TUNEL positive cells were determined.
Data are mean ±SEM of 3 separate experiments performed in triplicate.
*p < 0.05 versus medium alone.
Vol. 55, 2006 Neutrophil antimicrobial peptides and cell death 125
7A) with 50 µg/ml HNP1-3, a marked increase of TUNEL-
positive cells was observed as compared with medium-
treated cells. This increase was observed within 5 h and was
more pronounced after 24 h. Treatment of A549 cells with
30 µg/ml LL-37 also elicited increased TUNEL labeling af-
ter incubation for 5 and 24 h. Treatment with cytochalasin-D
was used as a positive control  and showed increased
TUNEL labeling (Fig. 7). Also in PBEC, both HNP1-3 and
LL-37 increased the number of TUNEL-positive cells, sug-
gesting that HNP1-3 and LL-37 also induce DNA breaks in
primary bronchial epithelial cells (Fig. 7B).
In this study, we analysed the mechanisms underlying the
induction of cell death by the antimicrobial peptides HNP1-3
and LL-37 in lung epithelial cells and Jurkat cells. In Jurkat
cells these mechanisms differed for HNP1-3 and LL-37:
while both peptides induced release of cytochrome c into
the cytosol, only HNP1-3 activated the cysteine proteases
caspase-3/-7 and caused cleavage of PARP. In addition,
while HNP1-3 increased PI/Annexin V double positive cells,
LL-37 increased PI positive cells without affecting Annexin
V. Furthermore, HNP1-3-induced cell death does not involve
Bcl-2 family members since HNP1-3 also induced cell death
in Bcl-2-transfected Jurkat cells. Finally, in lung epithelial
cells both HNP1-3 and LL-37 were shown to induce DNA
breaks and cytochrome c release, and neither HNP1-3 nor
LL-37 activated caspase-3/-7 in these cells.
Previous studies have provided limited insight into the
mechanisms involved in cell death induction by antimicro-
bial peptides. Neutrophil defensins have been demonstrated
to cause DNA breaks in leukemia cell lines, which is in line
with our observation that in epithelial cells neutrophil de-
fensins increase TUNEL labeling. Furthermore, the bovine
cathelicidin BMAP-28, which shares structural features with
LL-37: both are cationic peptides that are assumed to form
an a-helical peptide, was shown to induce cell death in nor-
mal proliferating human lymphocytes and human tumor cells
(including U937 and Jurkat cells) [32, 33], and the hCAP-
18-derived peptide hCAP-18109–136  was shown to induce
cell death in cells from the oral squamous cell carcinoma
cell line SAS-H1. In addition to LL-37, both BMAP28 and
hCAP-18109–136 affected mitochondria, induced DNA-breaks
and did not signifi cantly affect caspase activity, suggesting
that cell death induced by these cathelicidin-derived peptides
may be mediated via a (partly) shared mechanism.
Cell death may be mediated via either necrosis or a
programmed process called apoptosis. Caspases have been
shown to play a central role in apoptosis, which can be elic-
ited via an extrinsic death receptor-dependent, or via an in-
trinsic mitochondrial-mediated mechanism. Death receptors
are members of the TNF-receptor superfamily and are char-
acterized by an intracellular death domain, that transduces
the apoptotic signal via activation of caspase-8 and down-
stream caspases, resulting in apoptosis (reviewed in ). In
contrast to extrinsic apoptosis, receptor-independent apop-
tosis involves release of mitochondria-derived apoptogenic
factors into the cytosol, which activate caspase-9 and down-
stream caspases, resulting in apoptosis. Since HNP1-3, but
not LL-37 activates caspase-3/-7 in Jurkat cells, HNP1-3
may mediate cell death via apoptosis. However, while the
pancaspase inhibitor zVAD fully inhibited HNP1-3-, and
also etoposide-induced caspase-3/-7 activation, zVAD did
not affect defensin-induced cell death, and only had a minor
effect on etoposide-induced cell death. An explanation for
this discrepancy may be that inhibition of caspase activity
may result in necrotic cell death, as has been previously
reported . Another explanation may be an alternative,
initial caspase-independent pathway resulting in caspase-3/-
7 activation. Indeed, de Bruin et al.  demonstrated that
in etoposide-exposed melanoma cells zVAD did not cause
inhibition of PARP-cleavage. In contrast, the serine protease
inhibitor AEBSF was able to block caspase activation and
PARP cleavage, suggesting the involvement of a serine pro-
tease in the initiation of DNA-damage-induced cell death.
Members of the Bcl-2 family have been demonstrated to
regulate apoptosis, and various cell death stimuli have been
demonstrated to activate Bid or Bad, which are Bcl-2 family
members that promote cell death. These proteins may bind to
Bcl-2 family members localized in the mitochondrial mem-
brane, and include members that promote apoptosis (Bax
and Bak) and members that suppress apoptosis (Bcl-2 and
Bcl-XL) . In our study, we used Jurkat cells transfected
with anti-apoptotic Bcl-2 and showed that etoposide-induced
cell death was inhibited, while HNP1-3-induced cell death
was not affected in Bcl-2 transfected cells. This suggests that
neutrophil defensin-induced cell death is not mediated via
Bcl-2, but may be mediated via other members of the Bcl-2
family or via a direct effect on the mitochondrial membrane,
as has been suggested by Gera et al. .
Both HNP1-3 and LL-37 were shown to induce cy-
tochrome c release from mitochondria within 3 h after
stimulation. In contrast to studies suggesting that neutrophil
defensins enter the target cell while LL-37 sticks into the
cellular membrane [20, 23], this suggests that in addition
to HNP1-3, LL-37 may also penetrate the plasma mem-
brane and directly affect mitochondria. Direct activation
of mitochondria has been described for other peptides [38,
39]. Mastoparan, which is derived from wasp venom and
shares structural homology with LL-37, was the fi rst peptide
found to induce mitochondrial membrane permeabilisation
independent of upstream effectors , and subsequently,
many other amphipatic cationic alpha-helical peptides were
shown to affect the mitochondrial membrane, including
(KLAKLAK)2  and the human immunodefi ciency virus-
1-derived viral protein R (vpR) . Neutrophil defensins
and LL-37 may preferentially target the mitochondrial mem-
brane, since the composition of this membrane is similar to
that of prokaryotes, which are known to be more sensitive to
defensins than eukaryotic membranes .
The concentrations of HNP1-3 and LL-37 used in this
study may be relevant in vivo. HNP1-3 concentrations of
100 µg/ml have been demonstrated in purulent sputum from
chronic obstructive pulmonary disease  and cystic fi -
brosis patients , and plasma of sepsis patients has been
shown to contain up to 170 µg/ml HNP1-3 . LL-37
was demonstrated in the airways [9, 46], and concentra-
tions of about 20 µg/ml were shown in tracheal aspirates
of newborns, which were signifi cantly increased in infants
with pulmonary or systemic infections . Skin lesions
126 J. Aarbiou et al. Infl amm. res.
from patients with psoriasis, a condition associated with in-
creased LL-37 expression in the skin, were found to contain
LL-37 at a median concentration of 1360 µg/ml . Fur-
thermore, local high HNP1-3  and LL-37 levels may be
present in the sequestered environment between neutrophils
and their target cells. These data therefore suggest that the
concentrations used in our study may be relevant in vivo,
and that induction of cell death by antimicrobial peptides
What could be the biological relevance of the ability
of antimicrobial peptides to kill host cells? Activated neu-
trophils release antimicrobial peptides into the extracellular
environment, and this is likely to occur during antibody-de-
pendent cell cytotoxicity (ADCC), a mechanism that plays a
role in the clearance of virus-infected and transformed cells.
This may constitute an additional mechanism by which
neutrophil antimicrobial peptides contribute to host defense
against infection and against tumors. Whether these peptides
also contribute to the clearance of e. g. T cells by inducing
cell death, and thus contribute to the regulation of the adap-
tive immune response, requires further investigation. In
conclusion, the results from the present study show that anti-
microbial peptides induce cell death in airway epithelial cells
and in cells from the Jurkat T-cell line. In Jurkat cells, HNP1-
3 but not LL-37 induced caspase-3/-7 activation, suggesting
induction of apoptosis by HNP1-3 in these cells, while both
peptides induced cytochrome c release. In contrast to Jurkat
cells, neither HNP1-3 nor LL-37 induced caspase-3/-7 ac-
tivation in lung epithelial cells. The results from this study
suggest that the antimicrobial peptides HNP1-3 and LL-37
may induce cell death in eukaryotic cells via activation of
partly different intracellular pathways.
Acknowledgements. The authors would like to thank Jan Wouter
Drijfhout (Dept. of Immunohematology and Bloodtransfusion, LUMC,
Leiden, The Netherlands) for preparing synthetic LL-37, and Vicky
Alksnitis, Sylvia van der Meer, and Roberta Tse for their technical as-
sistance. Furthermore we would like to thank Peter Hordijk (Sanquin,
Amsterdam, The Netherlands) for helpful discussions and Jan-Paul
Medema (Dept. of Clinical Oncology, LUMC, Leiden, The Nether-
lands) and Jannie Borst (The Netherlands Cancer Institute, Amsterdam,
The Netherlands) for providing the Bcl-2-transfected Jurkat cells. This
study was supported by grants from the Netherlands Asthma Foundation
(grants 97.55 and 98.46) and the Netherlands Organization for Scientifi c
Research (NWO; grant 902-11-092).
 Bals R, Hiemstra PS. Innate immunity in the lung: how epithelial
cells fi ght against respiratory pathogens. Eur Respir J 2004; 23:
 Agerberth B, Charo J, Werr J, Olsson B, Idali F, Lindbom L et al.
The human antimicrobial and chemotactic peptides LL-37 and al-
pha-defensins are expressed by specifi c lymphocyte and monocyte
populations. Blood 2000; 96: 3086–93.
 Obata-Onai A, Hashimoto S, Onai N, Kurachi M, Nagai S, Shizuno
K et al. Comprehensive gene expression analysis of human NK
cells and CD8(+) T lymphocytes. Int Immunol 2002; 14: 1085–98.
Erratum: Int Immunol 2002; 14: 1469.
 Ganz T, Selsted ME, Lehrer RI. Defensins. Eur J Haematol 1990;
 Aarbiou J, Rabe KF, Hiemstra PS. Role of defensins in infl amma-
tory lung disease. Ann Med 2002; 34: 96–101.
 Soong LB, Ganz T, Ellison A, Caughey GH. Purifi cation and char-
acterization of defensins from cystic fi brosis sputum. Infl amm Res
1997; 46: 98–102.
 Gudmundsson GH, Agerberth B, Odeberg J, Bergman T, Olsson B,
Salcedo R. The human gene FALL39 and processing of the cathelin
precursor to the antibacterial peptide LL-37 in granulocytes. Eur J
Biochem 1996; 238: 325–32.
 Frohm NM, Sandstedt B, Sorensen O, Weber G, Borregaard N,
Stahle-Backdahl M. The human cationic antimicrobial protein
(hCAP18), a peptide antibiotic, is widely expressed in human sq-
uamous epithelia and colocalizes with interleukin-6. Infect Immun
1999; 67: 2561–6.
 Bals R, Wang X, Zasloff M, Wilson JM. The peptide antibiotic
LL-37/hCAP-18 is expressed in epithelia of the human lung where
it has broad antimicrobial activity at the airway surface. Proc Natl
Acad Sci USA 1998; 95: 9541–6.
 Frohm M, Agerberth B, Ahangari G, Stahle-Backdahl M, Liden S,
Wigzell H et al. The expression of the gene coding for the anti-
bacterial peptide LL-37 is induced in human keratinocytes during
infl ammatory disorders. J Biol Chem 1997; 272: 15258–63.
 Di Nardo A, Vitiello A, Gallo RL. Cutting edge: mast cell antimi-
crobial activity is mediated by expression of cathelicidin antimicro-
bial peptide. J Immunol 2003; 170: 2274–8.
 Sorensen OE, Follin P, Johnsen AH, Calafat J, Tjabringa GS,
Hiemstra PS et al. Human cathelicidin, hCAP-18, is processed
to the antimicrobial peptide LL-37 by extracellular cleavage with
proteinase 3. Blood 2001; 97: 3951–9.
 Sorensen OE, Gram L, Johnsen AH, Andersson E, Bangsboll S,
Tjabringa GS et al. Processing of seminal plasma hCAP-18 to
ALL-38 by gastricsin: a novel mechanism of generating antimicro-
bial peptides in vagina. J Biol Chem 2003; 278: 28540–6.
 Aarbiou J, Verhoosel RM, van Wetering S, de Boer WI, van
Krieken JHJM, Litvinov SV et al. Neutrophil defensins enhance
lung epithelial wound closure and mucin gene expression in vitro.
Am J Respir Cell Mol Biol 2003; 30: 193–201.
 Zanetti M. Cathelicidins, multifunctional peptides of the innate im-
munity. J Leukoc Biol 2004; 75: 39–48.
 Okrent DG, Lichtenstein AK, Ganz T. Direct cytotoxicity of
polymorphonuclear leukocyte granule proteins to human lung-
derived cells and endothelial cells. Am Rev Respir Dis 1990; 141:
 van Wetering S, Mannesse-Lazeroms SP, Dijkman JH, Hiemstra
PS. Effect of neutrophil serine proteinases and defensins on lung
epithelial cells: modulation of cytotoxicity and IL-8 production. J
Leukoc Biol 1997; 62: 217–26.
 Johansson J, Gudmundsson GH, Rottenberg ME, Berndt KD,
Agerberth B. Conformation-dependent Antibacterial Activity of
the Naturally Occurring Human Peptide LL-37. J Biol Chem 1998;
 Gera JF, Lichtenstein A. Human neutrophil peptide defensins in-
duce single strand DNA breaks in target cells. Cell Immunol 1991;
 Henzler Wildman KA, Lee DK, Ramamoorthy A. Mechanism of
lipid bilayer disruption by the human antimicrobial peptide, LL-37.
Biochemistry 2003; 42: 6545–58.
 Wimley WC, Selsted ME, White SH. Interactions between human
defensins and lipid bilayers: evidence for formation of multimeric
pores. Protein Sci 1994; 3: 1362–73.
 Oren Z, Lerman JC, Gudmundsson GH, Agerberth B, Shai Y.
Structure and organization of the human antimicrobial peptide LL-
37 in phospholipid membranes: relevance to the molecular basis for
its non-cell-selective activity. Biochem J 1999; 341: 501–13.
 Lichtenstein AK, Ganz T, Nguyen TM, Selsted ME, Lehrer RI.
Mechanism of target cytolysis by peptide defensins. Target cell
metabolic activities, possibly involving endocytosis, are crucial for
expression of cytotoxicity. J Immunol 1988; 140: 2686–94.
 Zaharatos GJ, He T, Lopez P, Yu W, Yu J, Zhang L. alpha-De-
fensins Released Into Stimulated CD8+ T-Cell Supernatants Are
Likely Derived From Residual Granulocytes Within the Irradiated
Allogeneic Peripheral Blood Mononuclear Cells Used as Feeders. J
Acquir Immune Defi c Syndr 2004; 36: 993–1005.
Vol. 55, 2006 Neutrophil antimicrobial peptides and cell death 127
 van Wetering S, Mannesse-Lazeroms SP, van Sterkenburg MA,
Daha MR, Dijkman JH, Hiemstra PS. Effect of defensins on in-
terleukin-8 synthesis in airway epithelial cells. Am J Physiol 1997;
 Werner AB, de Vries E, Tait SWG, Bontjer I, Borst J. Bcl-2 Fam-
ily Member Bfl -1/A1 Sequesters Truncated Bid to Inhibit Its Col-
laboration with Pro-apoptotic Bak or Bax. J Biol Chem 2002; 277:
 van Wetering S, van der Linden AC, van Sterkenburg MA, Rabe
KF, Schalkwijk J, Hiemstra PS. Regulation of secretory leukocyte
proteinase inhibitor (SLPI) production by human bronchial epithe-
lial cells: increase of cell-associated SLPI by neutrophil elastase. J
Investig Med 2000; 48: 359–66.
 Aarbiou J, Ertmann M, van Wetering S, Van Noort P, Rook D, Rabe
KF et al. Human neutrophil defensins induce lung epithelial cell
proliferation in vitro. J Leukoc Biol 2002; 72: 167–74.
 de Bruin EC, Meersma D, de Wilde J, den Otter I, Schipper EM,
Medema JP et al. A serine protease is involved in the initiation
of DNA damage-induced apoptosis. Cell Death Differ 2003; 10:
 Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM,
Pals ST, van Oers MH. Annexin V for fl ow cytometric detection
of phosphatidylserine expression on B cells undergoing apoptosis.
Blood 1994; 84: 1415–20.
 White SR, Williams P, Wojcik KR, Sun S, Hiemstra PS, Rabe KF
et al. Initiation of Apoptosis by Actin Cytoskeletal Derangement in
Human Airway Epithelial Cells. Am J Respir Cell Mol Biol 2001;
 Risso A, Zanetti M, Gennaro R. Cytotoxicity and apoptosis medi-
ated by two peptides of innate immunity. Cell Immunol 1998; 189:
 Risso A, Braidot E, Sordano MC, Vianello A, Macri F, Skerlavaj B
et al. BMAP-28, an Antibiotic Peptide of Innate Immunity, Induces
Cell Death through Opening of the Mitochondrial Permeability
Transition Pore. Mol Cell Biol 2002; 22: 1926–35.
 Okumura K, Itoh A, Isogai E, Hirose K, Hosokawa Y, Abiko Y et al.
C-terminal domain of human CAP18 antimicrobial peptide induces
apoptosis in oral squamous cell carcinoma SAS-H1 cells. Cancer
Lett 2004; 212: 185–94.
 Ashkenazi A, Dixit VM. Death Receptors: Signaling and Modula-
tion. Science 1998; 281: 1305–8.
 Fiers W, Beyaert R, Declercq W, Vandenabeele P. More than one
way to die: apoptosis, necrosis and reactive oxygen damage. Onco-
gene 1999; 18: 7719–30.
 Chittenden T. BH3 domains: intracellular death-ligands critical for
initiating apoptosis. Cancer Cell 2002; 2: 165–6.
 Chen LB. Mitochondrial membrane potential in living cells. Annu
Rev Cell Biol 1988; 4: 155–81.
 Modica-Napolitano JS, Brunelli BT, Koya K, Chen LB. Photoac-
tivation enhances the mitochondrial toxicity of the cationic rhoda-
cyanine MKT-077. Cancer Res 1998; 58: 71–5.
 Pfeiffer DR, Gudz TI, Novgorodov SA, Erdahl WL. The Peptide
Mastoparan Is a Potent Facilitator of the Mitochondrial Permeabil-
ity Transition. J Biol Chem 1995; 270: 4923–32.
 Ellerby HM, Arap W, Ellerby LM, Kain R, Andrusiak R, Rio GD et
al. Anti-cancer activity of targeted pro-apoptotic peptides. Nat Med
1999; 5: 1032–8.
 Jacotot E, Ravagnan L, Loeffl er M, Ferri KF, Vieira HLA, Zam-
zami N et al. The HIV-1 Viral Protein R Induces Apoptosis via a
Direct Effect on the Mitochondrial Permeability Transition Pore. J
Exp Med 2000; 191: 33–46.
 Lohner K, Latal A, Lehrer RI, Ganz T. Differential scanning mi-
crocalorimetry indicates that human defensin, HNP-2, interacts
specifi cally with biomembrane mimetic systems. Biochemistry
1997; 36: 1525–31.
 Panyutich AV, Hiemstra PS, van Wetering S, Ganz T. Human neu-
trophil defensin and serpins form complexes and inactivate each
other. Am J Respir Cell Mol Biol 1995; 12: 351–7.
 Panyutich AV, Panyutich EA, Krapivin VA, Baturevich EA, Ganz T.
Plasma defensin concentrations are elevated in patients with septi-
cemia or bacterial meningitis. J Lab Clin Med 1993; 122: 202–7.
 Agerberth B, Grunewald J, Castanos-Velez E, Olsson B, Jornvall
H, Wigzell H et al. Antibacterial components in bronchoalveolar
lavage fl uid from healthy individuals and sarcoidosis patients. Am
J Respir Crit Care Med 1999; 160: 283–90.
 Schaller-Bals S, Schulze A, Bals R. Increased levels of antimicro-
bial peptides in tracheal aspirates of newborn infants during infec-
tion. Am J Respir Crit Care Med 2002; 165: 992–5.
 Ong PY, Ohtake T, Brandt C, Strickland I, Boguniewicz M, Ganz
T et al. Endogenous antimicrobial peptides and skin infections in
atopic dermatitis. N Engl J Med 2002; 347: 1151–60.
 Ganz T. Extracellular release of antimicrobial defensins by human
polymorphonuclear leukocytes. Infect Immun 1987; 55: 568–71.