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IMMUNOBIOLOGY
From the Laboratory of Signal Transduction, Department
of Experimental Medicine and Biochemical Sciences, University of Rome
"Tor Vergata," Italy.
Dendritic cells (DCs) play a central role in the initiation and
regulation of the immune response. The modalities by which DCs are
committed to undergo apoptosis are poorly defined. Here it is shown
that, unlike death receptor ligands, UVB radiation triggers apoptosis
of human DCs very efficiently. UVB exposure is followed by the
activation of caspases 8, 9, and 3, by the loss of mitochondrial
transmembrane potential ( Most tissues are equipped with interstitial
dendritic cells (DCs) as an efficient alert system against foreign
antigens for the exquisite ability of DCs in capturing
macromolecules.1,2 Tissue-associated DCs are commonly
referred to as immature DCs (IDCs) because they lack some key surface
accessory molecules and are, therefore, unable to trigger T-cell
activation effectively. After antigen capture and processing, IDCs
undergo extensive morphologic and biochemical changes and migrate to
specialized lymphoid areas. Such mature DCs (MDCs) dismiss antigen
capture functions, become competent antigen-presenting cells (APCs),
and productively interact with T cells to initiate an antigen-specific
immune response.3
Two types of bone marrow-derived DCs colonize human skin from
peripheral blood to serve as professional APCs of environmental antigens. These are Langerhans cells (LCs), which reside mostly in the
basal and suprabasal layers of the epidermis, and dermal IDCs, which
are confined within the dermis.4-6 On antigen capture and
maturation, mature LCs and MDCs relocate from the skin to draining
lymph nodes in search of antigen-specific T cells.
Ultraviolet B (280-320 nm) radiation is a major stress-inducing agent
for most body surfaces. Exposure of the human skin to chronic and acute
UVB irradiation is known to cause immunosuppression.7 This
results largely from local effects, including both massive depletion of
LCs,8,9 likely due to apoptosis,10,11 and functional impairment in the costimulatory abilities of the residual LCs.12 UVB exposure may also suppress the T-cell
stimulatory capacity of human DCs.13 UV-induced
intracellular mediators such as ceramide, moreover, may profoundly
affect DC functions.14 Little is known, however, about
whether dermal human DCs trigger the apoptotic program under UVB
exposure or how they activate a protective response to UVB-induced
cellular stress.
Here we show that high-dose UVB radiation may induce very efficient
apoptosis of human DCs. This is associated with early mitochondrial
changes and is mediated by multiple caspase activation, resulting in
cytosolic and nuclear fragmentation. The up-regulation of FLIP and
bcl2, which occurs during DC maturation, may provide protection from
UVB-induced effects, conferring a survival advantage to MDCs.
In vitro culture of human dendritic cells
UVB irradiation
Evaluation of nuclear hypodiploidy and condensation Irradiated cells were washed in cold phosphate-buffered saline (PBS) and resuspended in hypotonic fluorochrome solution (50 µg/mL propidium iodide in 0.1% sodium citrate plus 0.1% Triton X-100), kept for 4 to 8 hours at 4°C in the dark, and analyzed by FACScan cytofluorometer (Becton Dickinson) for the evaluation of the percentage of the hypodiploid nuclei. Irradiated cells were incubated for 10 minutes at 37°C with 500 ng/mL Hoechst (Molecular Probes, Eugene, OR), and nuclear condensation was analyzed by fluorescence microscopy.Quantification of mitochondrial membrane permeability transition Cells (5 × 105/mL) were incubated for 15 minutes with 10 µg/mL JC1 (Molecular Probes). Cells were washed twice with cold PBS and immediately analyzed by flow cytometry. JC1 forms red fluorescent J-aggregates (590 nm) at higher  m and green
fluorescent monomers (527 nm) at low-membrane potential. Changes in
m were, therefore, evaluated by the shift in
fluorescence emission.
Western blotting Cells (2 × 106) were washed in PBS, and the pellet was resuspended in lysis buffer (150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 2% Triton X-100, 0.5% NP40) and protease inhibitors. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (Protran; Schleicher & Schuell, Keene, NH). Membranes were incubated with mouse mAb antihuman caspase 8 (clone 5F7; Upstate Biotechnology, Lake Placid, NY), rabbit polyclonal antihuman caspase 9 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse mAb antihuman caspase 3 (clone19; Transduction Laboratories, Lexington, KY), rabbit polyclonal antihuman-FLIP (kindly provided by Dr J. Tschopp, Institute of Biochemistry, University of Lausanne, Switzerland), mouse mAb antihuman FADD (clone 1; Transduction Laboratories), rabbit polyclonal antihuman bcl2 (Santa Cruz Biotechnology), rabbit polyclonal antihuman bclx-L (Santa Cruz Biotechnology), and rabbit polyclonal antihuman bax (Santa Cruz Biotechnology). For detection, secondary antibodies conjugated to horseradish peroxidase were used, followed by enhanced chemiluminescence (ECL; Amersham, Buckinghamshire, United Kingdom) and autoradiography.Poly(ADP-ribose) polymerase in vitro translation and cleavage assay Full-length human poly(ADP-ribose) polymerase (PARP) cDNA, cloned in Pgem vector, was used to synthesize [S35]methionine-labeled PARP by coupled T7 RNA polymerase-mediated transcription and translation in a reticulocyte lysate system (Promega, Madison, WI). Cell pellets were resuspended in 100 µL lysis buffer (50 mM NaCl, 2 mM MgCl2, 40 mM glycerophosphate, 5 mM EGTA, and 10 mM HEPES, pH 7.0). Cleavage reactions were performed in a volume of 36 µL containing 25 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 5 mM dithiothreitol, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/mL aprotinin, leupeptin, and pepstatin with 3 µL [35S]methionine-labeled PARP and 15 µL cell lysates. The reaction was incubated for 1 hour at 37°C. Samples were analyzed by SDS-PAGE. Gels were fixed (acetic acid 60%, methanol 40%, and glycerol 5%), treated with the Amplify solution (Amersham), and dried. Cleavage products were visualized by autoradiography.
UVB radiation induces apoptosis in human dendritic cells To establish whether DCs undergo apoptosis in response to UVB radiation, in vitro-cultured peripheral blood-derived DCs15 were exposed to doses of UVB ranging from 0.5 to 2.5 J/cm2. Approximately 75% of IDCs showed hypodiploid nuclei within 24 hours when exposed to 1 J/cm2 (2-minute exposure to a 9000 µW/cm2 source; peak emission, 313 nm) (Figure 1A). UVB doses less than 0.1 J/cm2 did not affect cell survival (not shown), but 1 J/cm2 UVB induced classical morphologic features of cellular apoptosis in IDCs, including nuclear fragmentation and formation of apoptotic bodies (see below). Only a minor proportion (less than 15%) of cells displayed a necrotic phenotype.
Variable levels of apoptosis can be induced in human DCs after cross-linking surface death receptors by ligands or agonistic antibodies.16-18 We observed that UVB-exposed IDCs trigger apoptosis efficiently in comparison with the approximately 15% to 20% nuclear hypodiploidy induced in 24 hours by optimal doses of soluble cross-linked FasL or TRAIL (Figure 1B). UVB-induced apoptosis of dendritic cells is largely mediated by caspase activation UVB-induced apoptosis may be triggered by both ligand-independent19,20 and ligand-dependent21,22 Fas clustering, with consequent caspase activation. The caspase cascade then mediates signal progression and proteolytic disassembling of apoptotic cells.We investigated the activation of caspase 8, caspase 9, and caspase 3 as key effectors responsible for the initiation, integration, and
effector phases of the caspase cascade in cells undergoing apoptosis.
Activation and processing of caspases 8, 9, and 3 could be detected in
IDCs as early as 2 hours after UVB exposure and was completed by 8 hours (Figure 2A-C). Caspase activity in
cell lysates derived from IDCs 4 hours after UVB treatment is fully competent in cleaving in vitro-translated PARP (Figure 2D).
To investigate the requirement for caspase activation in UVB-induced
apoptosis, IDCs were exposed to 1 J/cm2 UVB in the presence
of the general caspase inhibitor zVAD. Blocking zVAD-sensitive caspases
resulted in reduced UVB-induced nuclear fragmentation (Figure
3A) and in a substantial inhibition of
UVB-induced nuclear hypodiploidy in IDCs (Figure 3B), suggesting a
central role for caspases in the process.
UVB radiation induces m) from inner
membrane permeability transition is a key feature of the cellular apoptotic program and is often responsible for the amplification and
postmitochondrial progression of apoptotic signals.23 We therefore examined the induction of permeability transition in IDCs
exposed to 1 J/cm2 UVB. Disruption of m occurred in a
substantial proportion of IDCs after 4 to 6 hours (Figure 3C).
Interestingly, zVAD pretreatment could not inhibit UVB-induced m
loss, indicating that UVB exposure causes the induction of
mitochondrial inner membrane permeability transition in IDCs without
the involvement of caspases.
Mature dendritic cells are relatively resistant to UVB In vitro maturation of IDCs is promoted by cytokines and bacterial products and is associated with a variety of morphologic, structural, and functional changes that enable MDCs to act as fully competent APCs. We therefore asked whether the maturation of DCs could alter their UVB sensitivity. CD14 CD1a+ IDCs were cultured for
48 hours in the presence of lipopolysaccharide to induce maturation, as
assessed by the up-regulation of surface HLA-DR, CD80, CD86, and CD83
(Figure 4A). Then IDCs and MDCs from the
same donors were exposed to 1 J/cm2 UVB. MDCs displayed
significant resistance to UVB because nuclear hypodiploidy was not
detected until after 8 hours from UVB exposure, and it occurred with an
average delay of approximately 8 hours compared to IDCs
(Figure 4B).
Role for FLIP and bcl2 up-regulation in mature dendritic cells To gain insights into the molecular mechanisms mediating UVB resistance in MDCs, the kinetics of early signaling events that follow UVB exposure was investigated in further detail. Caspase 8 activation could not be detected in MDCs until 8 hours from UVB exposure, with a triggering delay of approximately 6 hours compared to IDCs from the same donors (Figure 5A). The activation of caspase 8 is controlled by endogenous FLIP at the death receptor signaling complex level.24 We, therefore, investigated the expression of FLIP during DC maturation. IDCs cultured for 24 hours in the presence of LPS showed approximately 10-fold up-regulation in FLIP expression levels compared to untreated IDCs from the same donor. The increase in FLIP expression occurred in MDCs without concomitant changes in FADD (Figure 5B) or caspase 8 expression (Figure 5A).
As with caspase 8, UVB-induced activation of caspases 9 and 3 was
significantly delayed in MDCs compared to IDCs (Figure
6A). Antiapoptotic members of the bcl2
family, such as bcl2 and bclxL, may negatively regulate the activation
of postmitochondrial caspases 9 and 3.25-27 We observed
that MDCs massively up-regulated bcl2, but not bclxL or bax, compared
to IDCs from the same donors (Figure 6B).
Delay in the execution of the apoptotic program finally appeared to
spare caspase-independent events, such as UV-induced mitochondrial changes, because the UVB-induced loss of
Here we show that human DCs execute a caspase-dependent apoptotic program in response to UVB irradiation. Importantly, the up-regulation of endogenous FLIP and bcl2 during maturation may delay UVB-induced caspase activation and confer a survival advantage to MDCs. Acute irradiation of body surfaces with UVB, which may occur after severe sunburn (0.5-5 J/cm2), results in transient but profound depletion of APCs that contributes to immunosuppression. Loss of LCs after human skin UVB irradiation might be attributed to apoptosis; apoptosis induction of LCs after in vitro or ex vivo UVB exposure has been reported.10,11 Murine LCs are sensitized by in vitro UVB exposure to undergo apoptosis after interaction with antigen and T cells.28 No information, however, is available about the effects of acute UVB irradiation on human dermal DCs. We report here that high-dose UVB irradiation triggers efficient apoptosis in human DCs. The apoptotic program includes multiple caspase activation, disruption of mitochondrial transmembrane potential, fragmentation of nuclear DNA, and apoptotic body formation. It has been proposed that UV light triggers protein synthesis-independent apoptosis by energy transfer and cell membrane perturbation, with consequent clustering of surface death receptors such as Fas.19,20,29 It has also been shown that homocellular Fas-FasL interactions contribute to UV-induced apoptosis in specific cell types.21,22 The latter mechanism seems not to play a major role in UVB-induced DC apoptosis; we observed that DCs display substantial resistance to FasL. On UV-induced receptor oligomerization, therefore, the cytosolic
adaptor FADD binds to clustered death domains and allows the
recruitment of caspase 8.19 This results in the
proteolytic autoactivation of caspase 8 and initiation of the caspase
cascade. We observed that UVB irradiation of DCs triggered the
activation of caspases 8, 9, and 3 within 2 hours. Accordingly, the
general caspase inhibitor zVAD could prevent most of the nuclear
features of the apoptotic response to UVB. Blocking caspase activation could not, however, prevent the induction of mitochondrial permeability transition observed in DCs after UVB exposure. Moreover, the delay in
caspase activation observed in MDCs compared to IDCs after UVB exposure
had no effect on the kinetics of mitochondrial changes induced by UVB
exposure. Together these results indicate that acute UVB irradiation
triggers 2 pathways in human DCs Cytokines or bacterial product-induced maturation of IDCs is accompanied by profound functional and structural changes. By up-regulating selected surface receptors, MDCs relocate from antigen-capturing areas to specialized lymphoid tissue and make productive contact with T lymphocytes. Here we show that DC maturation is associated with the acquisition of resistance to UVB irradiation and with the up-regulation of key antiapoptotic products, such as FLIP and bcl2. FLIP can compete with caspase 8 for binding to FADD, thus preventing caspase 8 clustering and autoactivation.30 Because the expression levels of caspase 8 and FADD do not change with maturation, the approximately 10-fold increase in FLIP is likely to be responsible for the delay (compared to IDCs) in caspase 8 activation observed in MDCs after UVB irradiation. The expression of FLIP is acquired during the differentiation of peripheral blood monocytes, conferring resistance to death ligands to both macrophages and IDCs.31-33 Most IDCs express relatively low levels of FLIP, which appear nevertheless sufficient to prevent caspase 8 activation after death receptor oligomerization by ligands. In this report we show that more dramatic environmental stressors, such as acute UVB irradiation, overcome the protective effects of FLIP in IDCs and result in efficient caspase 8 activation. However, further up-regulation of FLIP during DC maturation may confer additional resistance, perhaps critical for the survival of MDCs in vivo. Our data indicate, moreover, that UVB irradiation in DCs may damage
mitochondrial membranes by a caspase-independent pathway. This is
associated with a rapid dissipation of Coordinated FLIP and bcl2 induction during DC maturation, therefore, is likely to represent a key event in granting protection to UVB-induced cellular stress and underscores the need for prolonged survival of MDCs at sites of lymphocyte challenge and activation.
We thank Dr J. Tschopp (Institute of Biochemistry, University of Lausanne) for the anti-FLIP antibody and Dr Cristina Gagliardi (Department of Public Health, University of Tor Vergata) for help with flow cytometry. M.R.R. is a Fondazione Adriano Buzzati-Traverso fellowship holder.
Submitted August 4, 2000; accepted November 17, 2000.
Supported by Associazione Italiana Ricerca sul Cancro, Agenzia Spaziale Italiana, Consiglio Nazionale delle Ricerche Progetto Biotecnologie, Ministero dell' Universita' e della Ricerca Scientifica e Tecnologica, and European Commission Training and Mobility of Researchers Program.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Roberto Testi, Laboratory of Signal Transduction, Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata," via di Tor Vergata 135, 00133 Rome, Italy; e-mail: tesrob{at}flashnet.it.
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Top
Abstract
Introduction
-2-mercaptoethanol, 10% fetal bovine serum) in the presence of 50 ng/mL granulocyte-macrophage colony-stimulating factor (Mielogen 300;
Schering-Plough, Milan, Italy) and 5 ng/mL human recombinant IL-4 (R&D
Systems, Minneapolis, MN) and were cultured in 6-well plates (Costar;
Corning, Corning, NY) at the concentration of 5 × 105
cells/mL. Five-day culture cells (defined as IDCs) were then cultured
for an additional 48 hours in the presence of 400 ng/mL lipopolysaccharide (LPS; Sigma Chemical, St Louis, MO) to induce differentiation into MDCs. Purity and maturation of DCs was assessed by
immunostaining and FACS analysis with a FACScan (Becton Dickinson, San
Jose, CA), using fluorescein isothiocyanate-conjugated monoclonal antibodies (mAbs) to CD14, CD83, and CD80 and phycoerythrin-conjugated mAbs to CD1a, HLA-DR, and CD86 (Pharmingen, San Diego, CA).
and 
, untreated cells; 
and 
,
UVB-treated cells; 
and 
a caspase-dependent pathway
responsible for cytosolic and nuclear fragmentation and a
caspase-independent pathway directly targeting mitochondria.
.
J Exp Med.
1994;179:1109-1118