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ComparisonofbroadbandUVB,narrowband
UVB,broadbandUVAandUVA1onactivationof
apoptoticpathwaysinhumanperipheralblood
mononuclearcells
ArticleinPhotodermatologyPhotoimmunologyandPhotomedicine·March2007
DOI:10.1111/j.1600-0781.2007.00260.x·Source:PubMed
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Photodermatol Photoimmunol Photomed 2007; 23: 2–9
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Comparison of broadband UVB, narrowband UVB, broadband UVA and UVA1
on activation of apoptotic pathways in human peripheral blood mononuclear cells
Chanisada Tuchinda, Henry W. Lim, Faith M. Strickland, Esther A. Guzmán, Henry K. Wong
Department of Dermatology, Henry Ford Hospital, Detroit, MI, USA
Background/purpose: Ultraviolet (UV) radiation is an
important therapy for immune-mediated cutaneous
diseases. Activation of early apoptotic pathways may
play a role in the clinical effectiveness. Different UV
wavelengths have different efficacy for various diseases, but it remains unclear whether the ability to
induce apoptosis differs with respect to the wavelength,
and whether they induce apoptosis through the same
mechanism. The aim of this study is to analyze the
effects of different UV wavelengths that are used
clinically on normal human peripheral blood mononuclear cells (PBMCs).
Methods: PBMCs were treated with UV-light sources
broadband UVB, narrowband UVB, broadband UVA
and UVA1. Initiation of apoptosis was assessed by
flow cytometry by staining–treated cells for activated
caspases. Immunoblots were performed to measure for
cleaved caspase-3, -8, -9, cytochrome c, Bcl 2-interacting domain and poly-(ADP ribose) polymerase
cleavage.
Results: We demonstrate that all the UV radiation
sources induced caspase activation in a dose-and
time-dependent manner. Components of both the extrinsic and intrinsic pathways of apoptosis were activated by all of the UV wavelengths tested, but differed
in the level of energy needed for activation.
Conclusion: The greater effectiveness of UVB on
initiation of apoptotic pathway suggests that apoptosis
may play a role in the clinical efficacy of UVBresponsive inflammatory cutaneous diseases.
U
tributes to the effectiveness of phototherapy for inflammatory cutaneous diseases (1–6).
Apoptosis is a major process by which cells die.
Apoptosis is necessary for normal development and
maintenance of cellular homeostasis, as it is a mechanism to remove cells that are infected, damaged or no
longer needed. Defects in apoptosis can lead to autoimmune disease and/or cancer (7–9). Apoptosis is an
energy-dependent process characterized by hallmark
morphological features that include the condensation
of chromatin, DNA fragmentation and contortion of
plasma membrane into blebs and formation of membrane-bound apoptotic bodies (10).
Initiation of apoptosis is mediated by cysteine
aspartate-specific proteases (caspases). There are two
classes of caspases defined by their roles. Upstream or
initiator caspases (caspase-2, -8, -9, -10) propagate
death signals by activating downstream effector
caspases (caspase-3, -6, -7) in a cascade-like manner.
ltraviolet radiation (UV) has pleotropic effects
on human cells, both beneficial and deleterious.
Among its beneficial effects, UV radiation has been
shown to be an effective treatment for a wide variety
of immune-mediated cutaneous diseases, such as psoriasis, vitiligo and cutaneous T-cell lymphoma. Clinically, therapeutic spectra include broadband UVB
(BB-UVB; 280–320 nm), narrowband UVB (NBUVB; 311–312 nm), broadband UVA (BB-UVA;
320–400 nm) and UVA1 (340–400 nm). One of the
main effects of UV irradiation on human cells is the
induction of apoptosis (also called programmed cell
death). Thus, apoptosis is one mechanism that con-
Abbreviations: PBMCs, peripheral blood mononuclear cells;
UV, ultraviolet; UVA, ultraviolet A; BB-UVA, broadband
ultraviolet A; BB-UVB, broadband ultraviolet B; NB-UVB,
narrowband ultraviolet B; Bid, Bcl-2 interacting domain;
PARP, poly (ADP ribose) polymerase.
2
Keywords: apoptosis; caspase; lymphocytes; peripheral
blood mononuclear cells; ultraviolet light.
Comparison of different UV spectra in apoptotic pathways
The effector caspases then directly target cellular
protein structures, disrupt cellular metabolism, inactivate cell-death inhibitory proteins and activate additional destructive enzymes (11). In addition to the
caspases, many proapoptotic molecules and antiapoptotic proteins regulate apoptosis. The best-characterized regulators of apoptosis are members of the
Bcl-2 family such as Bax, Bad, Bid, Bik, Bim, Bcl-2
and Bcl-XL (12).
In caspase-dependent apoptosis, there are two main
pathways involved: the intrinsic pathway (mitochondrial/apoptosome pathway) and the extrinsic pathway
(death-receptor pathway). In the intrinsic pathway,
cells receiving apoptotic stimuli lose their mitochondrial membrane integrity, resulting in the release of
cytochrome c. Cytochrome c assembles with apoptosis
protease-activating factor 1 (Apaf-1) and procaspase9 to form an active apoptosome. Procaspase-9 is
activated and then activates downstream caspases.
The extrinsic pathway is regulated by members of
the tumor necrosis factor (TNF) superfamily, of which
six members are known so far, namely CD95 (Fas/
APO-1), TNF-receptor1 (TNF-R1), TNF receptorrelated apoptosis-mediating protein (TRAMP) receptor, TNF-related apoptosis-inducing ligand receptor1
(TRAIL-R1), TRAIL-R2 and death receptor 6 (DR6)
(13, 14). Binding of death receptors with their extracellular ligands results in the activation of molecules
containing motifs known as death domain such as
the Fas-associated death domain protein (FADD).
The death effector domain (DED) of FADD binds
to DED of initiator caspases (caspase-8 and -10) to
form a death-inducing signalling complex (DISC),
which results in activation of the initiator caspases.
Activated caspase-8 stimulates apoptosis via two
ways: (1) by direct cleavage and activation of caspase-3, and (2) in some cells activated caspase-8
cleaves Bid (Bcl-2 interacting domain) into truncated
Bid (tBid). Truncated Bid then translocates to the
mitochondria and promotes cytochrome c release,
which sequentially activates caspase-9 and-3 (15, 16),
linking the extrinsic and intrinsic pathways of apoptosis.
A number of studies have been performed to
evaluate the mechanism of UV-induced lymphocyte
or T-cell apoptosis. However, most were done in cell
lines established from animals or diseased patients
that have undergone many passages in the tissue
culture (17–21). Given the fact that apoptosis is a
cell-specific process, the effects of UV exposure on cell
lines might differ from those on normal human cells.
Furthermore, even among normal human cells, there
is likely a different response to treatment by UV
irradiation. In addition, although different wavelengths of UV radiation are effective in phototherapy,
it is unknown whether they differ in their ability to
induce apoptosis or whether they induce apoptosis
through different mechanisms. In this study, we compared and evaluated the effects of different UV
spectra (BB-UVB, NB-UVB, BB-UVA and UVA1)
on the activation of apoptosis in human peripheral
blood mononuclear cells (PBMCs).
Materials and methods
The analysis of human PBMCs was performed under
a minimal risk expedited protocol approved by The
Institutional Review Board of Henry Ford Hospital.
Human PBMCs from healthy volunteers were obtained from Red Cross donors.
Cell culture and isolation
PBMC was purified from red blood cells by centrifugation on a layer of Lymphoprept (Axis-Shield
PocAS, Oslo, Norway). After centrifugation, the
mononuclear cells’ interface was collected and red
blood cells were lysed using red blood cell lysis buffer
(0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA).
The cells were washed and maintained in RPMI1640. After UV radiation, PBMCs were resuspended
in RPMI-1640 supplemented with 100U/ml penicillin,
100 mg/ml streptomycin and 10% fetal bovine serum
and cultured at 37 1C in 5% CO2/95% aired-humidified atmosphere for 4, 16 or 24 h.
Reagents and antibodies
The following antibodies were used for Western blot
analysis: cleaved poly-(ADP ribose) polymerase cleavage (PARP) (Asp214) monoclonal antibody, cleaved
caspase-3 (Asp175) monoclonal antibody, cleaved caspase-8 (Asp384) monoclonal antibody, cleaved
caspase-9 (Asp330) monoclonal antibody, cytochrome
c antibody, Bid antibody (Cell Signaling Technology,
Beverly, MA, USA) and b-actin antibody (Santa Cruz
Biotechnology, Santa Cruz, CA, USA). The secondary
antibodies used were horse radish peroxidase (HRP)conjugated donkey anti-rabbit, HRP-conjugated goat
anti-mouse and HRP-conjugated mouse anti-goat
(Santa Cruz Biotechnology).
Radiation sources and dosimetry
Broadband UVB (BB-UVB) source: BB-UVB was
delivered using an FS40 lamp (National Biologics
Corp, Twinsburg, OH, USA) that has radiation spectra from 250 to 400 nm with a peak emission at
313 nm. FS40 lamp emits most of its energy within
3
Tuchinda et al.
the UVB range (61% UVB, 28.5% UVA2, 10%
UVA1 and 0.5% UVC). The UVB output was measured with an IL-1700 research radiometer (International Light, Newburyport, MA, USA) with an SED
240 detector and a UVB-1 filter. The output was
0.16 mW/cm2 at a tube to target distance of 17 cm,
which was the distance used in the irradiation in all
experiments.
Narrowband UVB (NB-UVB) source: NB-UVB was
delivered using eight bulbs of TL-01 lamps (National
Biological Corporation). The TL-01 lamp emits a
narrow spectrum of UVB (311–312 nm). The UVB
output was measured with IL-1700 research radiometer (International Light Inc., Newburyport, MA,
USA) with an SED 240 detector and a UVB-1 filter.
The output was 6 mW/cm2 at the tube to target
distance of 17 cm, which was the distance used in the
irradiation.
Broadband UVA (BB-UVA) source: UVA was delivered using two FS351 lamps (Q-Panel Lab Products, Cleveland, OH, USA). The lamp delivers
radiation spectra from 305 to 440 nm, with a peak
emission at 351 nm. The FS351 lamp emits most of its
energy in the UVA range (80.1% UVA1, 18.5%
UVA2 and 1.4% UVB). The UVA output was measured with an IL-1700 research radiometer (International Light Inc., Newburyport, MA, USA) with an
SED 033 detector and a UVA filter. The output was
2.7 mW/cm2 at the tube to target distance of 7.5 cm,
which was the distance used in the irradiation.
UVA1 source: UVA1 was delivered via Daavlin
SL3000 (Daavlin, Bryan, OH, USA). It delivers
UVA spectrum from 340 nm to 440 nm, with a peak
emission at 375 nm. The UVA1 output was measured
with a Daavlin X-97 Metre (Daavlin, Bryan, OH,
USA) and found to be 57 mW/cm2 at the tube to
target distance of 10 cm, which was the distance used
in the irradiation.
In vitro ultraviolet irradiation of PBMCs
PBMCs in colourless Hank’s solution were irradiated
with doses of BB-UVB (1, 2, 6, 10, 20 mJ/cm2), NBUVB (60 180 360 mJ/cm2) BB-UVA (0.3, 1.3, 2, 2.8 J/
cm2) and UVA1 (30, 50, 80 J/cm2). Mock UV-irradiated control cells were treated in an identical manner, except that the UV lamps were turned off. After
exposure to UV radiation, cells were kept in RPMI
medium supplemented with 100 U/ml penicillin,
100 mg/ml streptomycin and 10% foetal bovine serum
at 5% CO2 for the indicated periods of time before
further analysis by Western blot.
4
Detection of apoptotic cells
The number of apoptotic cells was determined by flow
cytometry analysis as the percentage of caspase-activated cells. In brief, 1 106 of PBMCs were exposed
to BB-UVB (1.5, 3, 10, 20, 30 mJ/cm2), NB-UVB (60,
180, 360 mJ/cm2) BB-UVA (0.3, 1.3, 2, 2.8 J/cm2) and
UVA1 (30, 50, 80 J/cm2). Irradiated PBMCs were
collected at 24 h after UV irradiation and incubated
with a CaspACEt FITC-VAD-FMK In Situ Marker
(Promega Corp., Madison, WI, USA) for 30 min.
PBMCs were then washed with Hank’s balanced salt
solution and immediately subjected to flow cytometry
analysis.
Western blot analysis
A total of 5 107 PBMCs per sample were washed in
Hank’s solution and lysed for 30 min on ice in 20 mM
HEPES-KOH, pH 7.9, 25% glycerol, 420 mM NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT,
0.2 mM PMSF, 1 mM sodium orthovanadate and
leupentin. After centrifugation at 10 000 g for
30 min, the amount of proteins in the supernatant
was determined by BCA protein Assay Reagent
(Pierce, Rockford, IL, USA) and BCA standard.
Ten micrograms of lysate was separated per lane on
10–15% SDS-PAGE and then electro-transferred to
polyvinylidene fluoride membranes. Nonspecific binding of membranes was blocked through 1-h incubation with I-blockt (Tropix, Bedford, IL, USA). The
membranes were incubated overnight with monoclonal antibody against PARP, cleaved caspase-3
monoclonal antibody, cleaved caspase-8 monoclonal
antibody, cleaved caspase-9 monoclonal antibody,
cytochrome c antibody and BID antibody. Bound
primary antibodies were reacted with anti-mouse
or anti-rabbit peroxidase-conjugate IgG for 1 h. To
monitor equal loading of proteins, membranes were
incubated with an antibody directed against b-actin
and re-incubated with mouse anti-goat HRP-conjugated IgG. An enhanced chemiluminescent substrate
system (Pierce) was used for detection.
Results
BB-UVB, NB-UVB, BB-UVA and UVA1 irradiation
induces apoptosis in a dose-dependent manner
To determine the sensitivity of human PBMCs to
apoptosis by UV irradiation, normal PBMCs were
purified and treated with the different wavelengths of
UV and measured for the activation of caspase to
determine the doses needed to initiate apoptosis. Only
UV irradiation was used and no additional sensitizers
were combined to study the effects of UV irradiation
Comparison of different UV spectra in apoptotic pathways
90
80
70
60
50
40
30
20
10
0
b
Pancaspases (+)
PBMCs (%)
Pancaspases (+)
PBMCs (%)
a
0
1.5
3
10
20
90
80
70
60
50
40
30
20
10
0
0
30
Dose (mJ/cm2)
d
90
80
70
60
50
40
30
20
10
0
Pancaspases (+)
PBMCs (%)
Pancaspases (+)
PBMCs (%)
c
0
0.3
1.3
2
60
180
360
Dose (mJ/cm2)
2.8
Dose (J/cm2)
90
80
70
60
50
40
30
20
10
0
0
30
50
80
Dose (J/cm2)
Fig. 1. Exposure to broad-band ultraviolet B (BB-UVB)-, narrow band ultraviolet (NB-UVB)-, BB-UVA- and
UVA1-induced caspase activation in peripheral blood mononuclear cells (PBMCs). PBMCs were incubated with
CaspACEt FITC-VAD-FMK in situ marker 24 h after irradiation and analyzed by flow cytometry as described in
materials and methods. Graphs show percentage of PBMC with activated caspases. (a) BB-UVB (1.5, 3, 10, 20,
30 mJ/cm2), (b) NB-UVB (60, 180, 360 mJ/cm2), (c) BB-UVA (0.3, 1.3, 2, 2.8 J/cm2), (d) UVA1 (30, 50, 80 J/cm2).
The data shown are representative of two independent experiments.
on cell properties. Caspase activation was measured
by a pan-caspase inhibitor detectible by fluorescence.
Activation of caspase by different UV sources in
PBMCs at 24 h after irradiation is shown in Fig. 1.
All the UV sources were capable of inducing caspase
activation in a dose-dependent manner.
During initiation of the apoptotic processes, PARP
enzyme is cleaved from 115 kDa into 89 and 24 kDa
products, and procaspase-3 is cleaved into 19 and
17 kDa products. To determine whether the caspase
activation leads to further sequential activation of
apoptotic cascade, we measured the level of cleaved
PARP and cleaved caspase-3 to determine the
extent to which UV-treated cells have activated
apoptotic pathway. Cells were treated with the
different UV light sources and harvested at 24 h
post-irradiation and analyzed by immunoblots using
antibodies to specific apoptotic proteins. The results
showed the cleavage of PARP (as demonstrated by the
89 kDa product), and the cleavage of caspase-3, with
all UV-treatment modalities (Fig. 2). The degree of
apoptosis measured by the activation of apoptotic
proteins is dependent on the UV dose. The lowest
doses of UV able to induce detectable cleavage
of caspase-3 were 1 mJ/cm2 for BB-UVB, 180 mJ/
cm2 for NB-UVB, 2 J/cm2 for BB-UVA and 30 J/cm2
for UVA1.
The UV dose-dependent caspase activation by flow
cytometry and immunoblot and the cleavage of PARP
indicates that all the spectra of tested UV sources
induced apoptosis in PBMCs.
BB-UVB, NB-UVB, BB-UVA and UVA1 induce
activation of the extrinsic and intrinsic pathways of
apoptosis
There are two major pathways that regulate apoptosis: the extrinsic and intrinsic pathways. To determine
whether the wavelengths of UV of each source activate the apoptotic pathways differently, activated
protein components of the intrinsic and extrinsic
pathways were analyzed. The intrinsic pathway of
apoptosis was analyzed using antibodies against cytochrome c and cleaved caspase-9. The activation of the
extrinsic pathway was analyzed using antibodies
against cleaved caspase-8 and Bid.
The immunoblot results showed that both the
intrinsic and extrinsic pathways of apoptosis could
be activated with all UV-treatment modalities. The
lowest dose that induced a release of cytochrome c or
cleaved caspase-9, indicators for intrinsic pathway
activation, was demonstrated to be 1, 60 mJ/cm2, 2
and 30 J/cm2 for BB-UVB, NB-UVB, BB-UVA and
UVA1, respectively (Fig. 3). The lowest dose that
induced cleavage of Bid or cleavage of caspase-8,
indicators of the extrinsic pathway activation, was 1,
60 mJ/cm2, 0.3 and 30 J/cm2 for BB-UVB, NB-UVB,
BB-UVA and UVA1, respectively (Fig. 4). The induc-
5
Tuchinda et al.
BB-UVB
(mJ/cm2)
0
1
NB-UVB
(mJ/cm2)
6 10 20
2
BB-UVA
(J/cm2)
0 60 180 360
UVA1
(J/cm2)
0 0.3 1.3 2 2.8
0
30
50
80
Full length (115 kDa)
Cleaved PARP
(89 kDa)
PARP
Caspase-3
(19/17 kDa)
Cleaved caspase-3
(19/17kDa)
Actin
(42 kDa)
42kDa
Fig. 2. Broad-band ultraviolet B (BB-UVB)-, narrow band ultraviolet B (NB-UVB)-, BB-UVA- and UVA1induced cleavage of poly-(ADP ribose) polymerase (PARP) and caspase-3 in peripheral blood mononuclear cells
(PBMCs). PBMCs were treated with BB-UVB (1, 2, 6, 10, 20 mJ/cm2), NB-UVB (60, 180, 360 mJ/cm2), BB-UVA
(0.3, 1.3, 2, 2.8 J/cm2) and UVA1 (30, 50, 80 J/cm2). After 24 h, the treated cells were lysed and Western blot was
performed using specific antibodies against full-length PARP (115 kDa) cleaved PARP (89 kDa) and cleaved
caspase-3 (19, 17 kDa). An equal level of protein was analyzed as shown by the level of actin (42 kDa).
NB-UVB
(mJ/cm2)
BB-UVB
(mJ/cm2)
Cytochrome c
(14kDa)
Cleaved caspase-9
(37kDa)
Actin
(42kDa)
0
1
0
2 6 10 20
60 180 360
1
2
0
6 10 20
60 180 360
37kDa
0 1
2 6 10 20
0
50
80
14kDa
0 0.3 1.3 2 2.8
0
30 50
80
37kDa
0 0.3 1.3 2 2.8
0 60 180 360
30
14kDa
37kDa
42kDa
UVA1
(J/cm2)
0 0.3 1.3 2 2.8
14kDa
14kDa
0
UVA
(J/cm2)
42kDa
37kDa
0
42kDa
30 50
80
42kDa
Fig. 3. Broad-band ultraviolet B (BB-UVB)-, narrow-band ultraviolet A (NB-UVB)-, BB-UVA- and UVA1induced activation of the intrinsic pathway of apoptosis. Peripheral blood mononuclear cells (PBMCs) were
treated with BB-UVB (1, 2, 6, 10, 20 mJ/cm2), NB-UVB (60, 180, 360 mJ/cm2), BB-UVA (0.3, 1.3, 2, 2.8 J/cm2) and
UVA1 (30, 50, 80 J/cm2). After 24 h, the cells were lysed and Western blot was performed using antibodies against
cytosolic cytochrome c (14 kDa), and cleaved caspase-9 (37 kDa). The level of actin (42 kDa) is shown.
tion of intrinsic and extrinsic pathway of apoptosis
was UV dose dependent.
BB-UVB-, NB-UVB-, BB-UVA- and UVA1-induced
apoptosis is dependent on duration post-irradiation
To investigate the duration necessary to detect the
apoptotic process after UV irradiation, PBMCs were
UV treated and protein extract was harvested after
different times. PBMCs were irradiated with 3 mJ/cm2
for BB-UVB, 180 mJ/cm2 for NB-UVB, 2.8 J/cm2 for
BB-UVA and 50 mJ/cm2 for UVA1. Cells were harvested at 4, 16 and 24 h after irradiation and analyzed.
At 4 h, activation of caspase-3 and cleavage of PARP
was detectable in PBMCs treated with 180, 2.8 or 50 J/
cm2 of NB-UVB, BB-UVA, and UVA1, respectively.
6
Before 4 h, there was minimal activation of components of apoptosis. Apoptosis was observed clearly at
16 h after irradiation irrespective of the UV sources
(Fig. 5). There was a progressive increase in the level
of activated apoptotic proteins, indicating that apoptotic proteins were continually generated until 24 h
after UV treatment.
Discussion
Ultraviolet-induced apoptosis is likely a mechanism of
action that contributes to the clinical properties of
phototherapy in the treatment of immune-mediated
cutaneous diseases. A number of studies have
been performed to evaluate the molecular effects of
UV-induced apoptosis, but these studies have been
Comparison of different UV spectra in apoptotic pathways
NB-UVB
(mJ/cm2)
BB-UVB
(mJ/cm2)
0
Full length Bid
(22kDa)
Cleaved Bid
(15kDa)
1
2
0 60 180 360
6 10 20
22kDa
15kDa
Full length
caspase-8 (57kDa)
Cleaved caspase-8
(43xDa)
0
1
2
0
6 10 20
0
1
2
6 10 20
0 0.3 1.3
2 2.8
0
30
50
80
22kDa
22kDa
15kDa
15kDa
15kDa
0 0.3 1.3 2 2.8
0
30 50 80
57kDa
43kDa
57kDa
43kDa
57kDa
43kDa
0
0 0.3 1.3 2 2.8
0 60 180 360
42kDa
42kDa
UVA1
(J/cm2)
22kDa
60 180 360
57kDa
43kDa
Actin
(42kDa)
UVA
(J/cm2)
30 50 80
42kDa
42kDa
Fig. 4. Broad-band ultraviolet B (BB-UVB)-, narrow-band ultraviolet A (NB-UVB)-, BB-UVA- and UVA1
induced activation of the extrinsic pathway of apoptosis. Peripheral blood mononuclear cells (PBMCs) were
treated with Broad-band ultraviolet B (BB-UVB) (1, 2, 6, 10, 20 mJ/cm2), NB-UVB (60, 180, 360 mJ/cm2), BBUVA (0, 0.3, 1.3, 2, 2.8 J/cm2) and UVA1 (30, 50, 80 J/cm2). After 24 h, cell extracts were prepared and Western
blot was performed using antibodies to detect caspase-8 cleavage (full-length 57 kDa, cleaved caspase-8 43 kDa),
Bid cleavage (full-length 22 kDa, cleaved Bid 15 kDa) and actin (42 kDa).
BB-UVB
(3 mJ/cm2)
Time (h)
0
4
16
Time (h)
24
0
4
16
UVA1
(50 J/cm2)
BB-UVA
(2.8 J/cm2)
NB-UVB
(180 mJ/cm2)
Time (h)
24
Caspase-3
PARP
0
4
16
Time (h)
24
0
4
16
24
Cleaved
Caspase-3
Full length
Cleaved
PARP
Actin
Fig. 5. Time-course study for ultraviolet (UV)-induced apoptosis in peripheral blood mononuclear cells (PBMCs).
PBMCs were treated with Broad-band ultraviolet B(BB-UVB) (3 mJ/cm2), narrow-band ultraviolet B (NB-UVB)
(180 mJ/cm2), BB-UVA (2.8 J/cm2) and UVA1 (50 J/cm2). After different incubation periods (4, 16 and 24 h),
Western blot was performed against full-length poly-(ADP ribose) polymerase (PARP) (115 kDa), cleaved PARP
(89 kDa), cleaved caspase-3 (19, 17 kDa) and actin (42 kDa).
performed in cell lines (17, 21, 22). The goal of this
study is to determine the effects of different UV
wavelengths in freshly isolated primary PBMCs from
healthy donors to gain an insight into the properties
of the different wavelengths. We compared sources
of UV that are currently available in the treatment of
cutaneous diseases to better understand the difference
in how the different wavelengths may act on signaling
pathways in normal PBMCs, which may more accu-
rately reflect how primary cells in vivo may be affected
by phototherapy. With respect to PBMCs, we show
here that all the different sources of UV used in our
study are able to induce apoptosis. Pathways involved
in apoptosis from both the intrinsic and extrinsic
pathways are activated by all sources of UV; however,
the energy level required differed.
Table 1 summarizes the minimal dose of each UV
spectrum needed in the initiation of the apoptotic
7
Tuchinda et al.
Table 1. Minimal doses of energy needed for activation
Western blot
BB-UVB
(mJ/cm2)
NB-UVB
(mJ/cm2)
BB-UVA
(J/cm2)
UVA1
(J/cm2)
Cytochrome c
Cleaved caspase-9
Caspase-8 cleavage
Bid cleavage
1
2
1
1
60
60
60
60
2
2
2
1.3
50
30
30
30
pathway. UVB showed a higher potency in initiation
of apoptosis in comparison with UVA. The minimal
dose required for induction of apoptosis by BB-UVB
irradiation was 1 mJ/cm2 as determined by Western
blot. This is similar to a study by Caricchio et al.
(23)which found that a dose as low as 1 mJ/cm2 of
UVB can induce apoptosis in human lymphocytes .
Our results are in agreement with the study of
Breuckmann et al. (24) which demonstrated that
UVB-induced extrinsic apoptosis in human T cells.
However, we detected a higher level of apoptotic cells
than reported in their study at the same dose of UVB
irradiation. This discrepancy might be explained by
the fact that different cell types and different radiation
sources were used in these two studies.
It has been reported that UVA and UVB are able to
induce both the intrinsic and extrinsic pathways of
apoptosis (2, 21, 22, 23, 25–28). Our findings show
that all wavelengths of UV tested have the ability to
activate effectively both the intrinsic and extrinsic
pathway of apoptosis in PBMCs. Because of the
ability of both pathways to feedback and activate
the other apoptotic pathway in some cells, there is the
possibility that one pathway is preferentially activated
by a certain wavelength, and the activation of the
other pathway is a secondary effect. This possibility
remains to be elucidated.
Phototherapies such as NB-UVB and 308 nm-excimer laser have been reported to induce T-cell apoptosis in psoriatic lesions (29, 30). However, the
mechanism by which these therapies induced apoptosis has not been clearly defined. In our present study,
we show that NB-UVB has the ability to induce
apoptosis by activation of both the intrinsic and
extrinsic pathways of apoptosis at doses clinically
relevant for phototherapy. We began to detect apoptosis in PBMCs at the lowest dose (60 mJ/cm2), with a
significant level of apoptosis at doses of 180 and
360 mJ/cm2. Levels of apoptosis induced by NBUVB in vitro differed from a study by Ozawa et al.
(6) because Annexin V/PI staining was used to detect
apoptotic cells in their study. Furthermore, they
studied the CD31-enriched T-cell population. Similar
to our results, the level of apoptosis in their study was
also UV dose- and post-irradiated-time dependent.
8
UVA- and UVA1-induced apoptosis in PBMCs
showed activation of both the intrinsic and extrinsic
pathways, which is in agreement with previous studies
(2, 17, 21). The level of energy needed to initiate
apoptotic signaling is higher for UVA than UVB. In
the studies presented, the UV sources emitting a more
focused spectrum (narrow-band) of wavelengths required a greater level of energy to induce apoptosis.
This is consistent with the observation that a higher
level of energy is necessary in the narrowband spectra
of UVA (UVA-1) and UVB (NB-UVB) in treatment.
In summary, our results show that all UV sources
studied are capable of inducing apoptosis in normal
PBMC at clinically relevant dose ranges (Table 1).
However, UVB induces apoptosis with a greater
efficiency than UVA. UVB is clinically effective in
the treatment of psoriasis, mycosis fungoides and
other T-cell inflammatory diseases, whereas UVA is
combined with psoralen to enhance efficacy, suggesting that the increased sensitivity to apoptosis may be a
mechanism for clinical effectiveness. The findings may
provide a baseline for understanding how the different
clinically important UV therapies affect PBMC, and
incorporating these properties will be important optimizing phototherapy in the treatment of cutaneous
diseases.
Acknowledgements
This study was supported by a grant from Faculty of
Medicine Siriraj Hospital, Bangkok, Thailand to CT,
NIH grant KO8-AR47818 and Dermatology Foundation Clinical Career Development Award to H.K.W.,
and NIH grant R01-AR47951 and Henry Ford Health
System investigator award to F.M.S.
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Accepted for publication 25 August 2006
Corresponding author:
Henry K. Wong, M.D., PhD
Department of Dermatology
Henry Ford Health System
One Ford Place, 4D
Detroit, MI, USA
Tel: 1313-874-9171
Fax: 1313-874-4851
e-mail: [email protected]
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