| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, Department of Dermatology, University Münster, Münster, Germany; and
The Wistar Institute, Philadelphia, PA 19104
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
There is increasing evidence that apoptosis plays an important role in the homeostasis of the immune system (7). The Fas Ag (APO-1, CD95) is a type I membrane protein (8, 9) that mediates apoptotic cell death upon interaction with its natural ligand, Fas ligand (FasL) (CD95L) (10). Mutation of the fas or fasL gene causes abnormal lymphoproliferation and generalized autoimmune disease in lpr and gld mice, respectively (11, 12). Recently, it was shown that the Fas/FasL system is crucial for the existence of immune-privileged organs such as the testis (13). Testis grafts from mice expressing FasL survive indefinitely when transplanted into allogeneic animals, while testis derived from gld mice lacking functional FasL are rejected. There is also recent evidence for a relationship between Fas/FasL-mediated cell death and tolerance induction following Ag injection into the anterior chamber of the eye (14), another immune-privileged organ which expresses high levels of FasL (15).
Since tolerance can be considered a form of immune privilege, we addressed whether UV-induced tolerance is mediated via the Fas/FasL system. Here, we show that 1) lpr and gld mice that lack expression of functional Fas and FasL, respectively, are resistant to UV-induced tolerance; 2) adoptive transfer experiments revealed that Fas and FasL expression is crucial for development of UV-mediated tolerance in the recipient but not in the donor; 3) T cells from UV-tolerized donors induce the cell death of hapten-pulsed bone marrow (BM)-derived dendritic cells (DC); 4) DC obtained from lpr and gld mice are resistant to cell death induced by T suppressor cells; and 5) the addition of IL-12, which was recently found to break UV-induced tolerance (16, 17, 18), can rescue DC from cell death induced by either suppressor cells or rFasL. Taken together, these data provide evidence for an important link between the apoptosis-associated Fas/FasL system and UV-induced immune tolerance. Furthermore, they demonstrate for the first time that T suppressor cells exert their suppressive activity via the Fas/FasL system and show that IL-12 might break tolerance by interfering with the Fas/FasL system.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
For detection of Fas expression, a rabbit polyclonal IgG (M-20,
Santa Cruz Biotechnology, Santa Cruz, CA) was used. FasL was detected
using an FITC-conjugated anti-FasL mAb (Alexis, Grünberg,
Germany). Murine rIFN-
was obtained from Genzmye (Cambridge, MA),
rFasL was kindly provided by K. Schulze-Osthoff (Department of Internal
Medicine, University of Tübingen, Tübingen, Germany), and
murine rIL-12 was a generous gift from S. Wolf (Genetics Institute,
Cambridge, MA).
Contact hypersensitivity
C3H/HeN, C3H/HeN-lpr, and C3H/HeN-gld mice were obtained from Bomholtgard Breeding and Research Center (Ry, Denmark). Mice at an age of 8 to 10 wk were used for the experiments. Mice (referred to as C3H, lpr, and gld, respectively) were sensitized on day 0 by painting 25 µl of 0.5% 2,4-dinitrofluorobenzene (DNFB) onto the shaved back. After 5 days, mice were challenged by painting 20 µl of 0.3% DNFB onto the left ear. Ear swelling was quantitated 24 h later using a spring-loaded micrometer. CHS was determined as the amount of swelling of the hapten-challenged ear compared with the thickness of the vehicle-treated ear in sensitized animals and expressed in centimeters x 10-3 (mean ± SD). After 2 weeks, mice were resensitized through nonirradiated abdominal skin and challenged on the right ear after 5 days. Data were analyzed by Student’s t test, and differences were considered significant at p < 0.05. Each group consisted of at least seven mice, and each experiment was performed at least three times.
UV irradiation
Mice were exposed to 1000 J/min UV on the shaved back daily for 4 consecutive days. FS-20 fluorescent lamps (Westinghouse Electric, Pittsburgh, PA), which emit most of their energy within the UVB range (290–320 nm), were used. DNFB was applied to the surface of the irradiated area 24 h after the last UV exposure.
Adoptive transfer
Donor mice were exposed to UV and sensitized with DNFB through UV-exposed skin. At 10 days after sensitization, spleens and regional lymph nodes were removed, and single-cell suspensions were prepared. The cell number was adjusted to 2.5 x 108 cells/ml, and 200 µl were injected i.v. into naive recipient mice. Recipients were sensitized 24 h later by epicutaneous application of DNFB on the shaved back. After 5 days, mice were challenged on the left ear, and ear swelling was evaluated 24 h later. For control purposes, identical numbers of cells obtained from untreated or DNFB sensitized mice were injected.
Isolation of T cells
Peripheral lymph nodes were obtained, single cell suspensions were prepared, and lymphocytes were depleted of RBCs. T cells were purified using a two step procedure consisting of a nylon wool column passage and the subsequent depletion of remaining contaminating cells with Ab-coated glass columns (Cellect Mouse T cell kit, Tebu, Edmonton, Canada), resulting in a >99% pure T cell preparation.
Generation of DC
DC were generated by culture of BM cells as previously described (19), although with some modifications. BM was collected from tibias, erythrocytes were lysed, and the remaining cells were cultured in petri dishes at a density of 0.5 x 106 cells/cm2 for 4 h. Nonadherent cells were collected, and 1 x 106 cells/ml were placed in 24-well plates in RPMI 1640 medium (5% FCS, 50 µM 2-ME, 1% nonessential amino acids, 20 µg/ml gentamicin), 150 U/ml granulocyte macrophage CSF, and 100 U/ml IL-4. Two-thirds of the medium was replaced every 2 days, and nonadherent cells were harvested on days 7 or 8. DC aggregates were purified by 1 g sedimentation and subcultured in 6-well plates. The expression of surface molecules characteristic for DC was determined by flow cytometry. DC cultures of >70% brightly I-A+ cells were used.
Flow cytometry
Aliquots of 1 x 105 DC were incubated with mAbs against I-Ab,d,q, I-Ed,k (M5/114), B7-1 (1G10), B7-2 (Gl-1) (PharMingen, San Diego, CA), CD11c (N418) (Endogen, Boston, MA), Thy-1.2 (30 H-12) (American Type Culture Collection, Rockville, MD), and normal rat IgG2b (PharMingen) as isotype controls for 45 min on ice. After washing, cells were incubated with FITC-conjugated goat anti-rat IgG (Boehringer Mannheim, Mannheim, Germany). Propidium iodide (PI) was added, and cells were washed twice and subsequently analyzed in a flow cytometer (Epics XL, Coulter, Miami, FL).
Coculture of DC and lymphocytes
DC (1 x 106) were incubated for 48 h with 5 x 106 purified T cells from the regional lymph nodes of sensitized, UV-tolerized (i.e., UV-exposed and DNFB-sensitized), or naive mice. Cultures were supplemented with 2,4-dinitrobenzenesulfonic sodium salt (0.1 mM, DNBS). In some cultures, rIL-12 (200 ng/ml) was added at the start of coculture. Cells were harvested and assessed by flow cytometry. DC were identified in forward light scatter (FSC)/side light scatter (SSC) dot plots by their larger size and granularity, and the DC- or T cell-specific gates were subsequently set. Cell viability was evaluated by flow cytometry using PI incorporation and scatter characteristics (lower FSC).
Detection of cell death
The murine DC line XS52 (kindly provided from A. Takashima, Department of Dermatology, University of Dallas, Dallas, TX) (20) was treated with 50 ng/ml of human rFasL. Cells were evaluated for apoptosis using a cell death detection ELISA (Cell Death Detection ELISAPLUS, Boehringer Mannheim) 16 h later. The basis of this test is the detection of mono- and oligonucleosomes in the cytoplasmic fractions of cell lysates using biotinylated anti-histone- and peroxidase-coupled anti-DNA Abs. The amount of nucleosomes is photometrically quantified by the peroxidase activity retained in the immunocomplexes.
Northern blot analysis
Cells (2 x 105) were washed twice with PBS, lysed in 4 M guanidine thiocyanate (0.5 ml)/300 mM sodium acetate (pH 4.8)/1% lauroylsarcosine/100 mM 2-ME for 10 min, vortexed for 1 min, and frozen at -20°C until used. Cell lysates were extracted once with acidic phenol (pH 4.8) and twice with chloroform. Precipitation was performed with equal volumes of isopropanol and centrifugation at 14,000 revolutions per minute for 10 min. The pellet was rinsed with 70% ethanol, residual solvent was evaporated in a vacuum desiccator, and the pellet was dissolved in double distilled water. The amount of total RNA was determined photometrically at 260 nm. For Northern blot analysis, total cellular RNA was denatured in 50% formamide, 6% formaldehyde, and 1x MOPS (0.002 M 3-(N-morpholino)propane-sulfonic acid, 0.05 M sodium acetate (pH 7.0), and 0.01 M Na2/EDTA) at 65°C for 5 min. Total RNA (10 µg) was separated by gel electrophoresis using 1% agarose gels containing 2.2% formaldehyde and 1x MOPS. Gels were blotted onto nylon membranes (Hybond-N, Amersham, Buckinghamshire, U.K.) and baked at 80°C for 2 h. Membranes were then prehybridized in 50% formamide, 10% dextran sulfate, 1% SDS, and 1 M NaCl for at least 15 min. Hybridization was conducted using deoxyadenosine-5'-[32P]triphosphate-labeled cDNA probes encoding murine Fas (kindly provided by S. Nagata, Department of Genetics, Osaka University, Osaka, Japan) at 42°C for at least 12 h in a hybridization oven (Hybaid, London, U.K.). After hybridization, filters were washed in 2x SSC at room temperature for 10 min, in 2x SSC containing 1% SDS at 60°C for 1 h, and finally in 0.1x SSC for 1 h. Filters were exposed to x-ray films at -70°C.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To address whether UV-induced tolerance is mediated via the
Fas/FasL system, we used lpr and gld mice, which
are defective in functional Fas and FasL expression, respectively (11, 12). For better comparison with other data, a genetic background was
chosen which is known to be UV-susceptible and which is routinely used
in photoimmunologic studies (C3H/HeN) (21). UV-induced
immunosuppression was analyzed by determining CHS responses after
sensitization with the contact sensitizer DNFB through UV-exposed skin.
Both lpr and gld mice exhibited a profound
UV-induced suppression of CHS that was indistinguishable from that of
C3H mice (Fig. 1
A).
UV-induced specific immune tolerance was analyzed by resensitizing the
mice through non-UV-irradiated skin 2 wk after the initial UV exposure.
Whereas C3H mice became tolerant to the hapten DNFB, neither
lpr nor gld mice developed tolerance (Fig. 1B). These data support the hypothesis that the
Fas/FasL system is essential for the generation of UV-induced
tolerance. Furthermore, they indicate that the suppression of CHS and
the induction of tolerance by UV light are separate events and mediated
by different pathways; this observation supports previous findings
(22).
|
Tolerance can be transferred to naive, nonirradiated mice by
adoptive transfer of T cells from tolerized mice, indicating the
existence of hapten-specific T suppressor cells (6). To test the role
of Fas/FasL in suppressor cell function, we performed adoptive transfer
experiments using different combinations of C3H, lpr, and
gld mice as donors or recipients. The transfer of lymph node
and spleen cells from UV-exposed and DNFB-treated C3H mice into naive
C3H mice inhibited the subsequent DNFB sensitization of the recipients
(Fig. 2A), confirming
the presence of suppressor cells within the transferred cell
population. In contrast, the transfer of cells from UV-exposed and
hapten-sensitized lpr donors into naive lpr mice
did not inhibit subsequent DNFB sensitization; similar findings were
obtained with gld mice (data not shown). To determine
whether the expression of Fas and FasL, respectively, by the recipient
or by donor-derived cells is required in mediating tolerance,
cross-transfer experiments were performed. Suppression was observed
when cells from UV-exposed and hapten-treated lpr donors
were injected into naive C3H mice (Fig. 2B), and also
when cells from UV-exposed and hapten-treated gld mice were
transferred into naive C3H mice (Fig. 2D). However,
both naive lpr and naive gld recipients of T
suppressor cells from UV-exposed and hapten-treated C3H mice exhibited
normal sensitization against the specific hapten (Fig. 2, C
and E). Together, these findings show that the
transfer of tolerance does not require Fas or FasL expression on the
suppressor cells but does require both molecules on the cells of the
recipient.
|
The present findings indicate that the Fas/FasL system is
essential for the mediation of UV-induced tolerance but not for the
generation of those T cells transferring suppression. Thus, one might
suspect that these T cells mediate tolerance by killing immunocompetent
cells which are important during (re)sensitization. However, since the
transfer experiments (Fig. 2
) clearly showed that the transfer of
tolerance does not require Fas or FasL expression on the suppressor
cells but does require both molecules on the cells of the recipient, we
surmised that T cells from UV-tolerized mice might not kill their
potential targets via the expression of FasL, but instead may induce
recipient cells that are critical during sensitization to undergo cell
death in a Fas/FasL-dependent manner. Thus, we hypothesized that APCs
may undergo apoptosis when presenting the hapten to T suppressor cells
and that this action is mediated via the Fas/FasL system. To test this
hypothesis, coincubation experiments of DC and T suppressor cells were
performed. However, since UV-induced T suppressor cells have not been
phenotypically characterized, and specific markers do not exist, it was
obligatory to use bulk T cell suspensions from UV-tolerized (i.e.,
UV-exposed and DNFB-sensitized) mice as a source for T suppressor
cells. Therefore, it is important to note that the term "T suppressor
cells" used throughout this manuscript refers to such bulk T cell
suspensions. T cells from naive, DNFB-sensitized, or UV-tolerized C3H
mice were coincubated with syngeneic BM-derived DC in the presence of
the water soluble DNFB-analogue, DNBS, for 48 h. Cell viability
was evaluated by flow cytometry using PI incorporation and scatter
characteristics (Fig. 3 shows a
representative FACS profile). In the absence of growth factors, DC
gradually die in culture, and this death is largely independent of
coculture with T cells in the absence of hapten. However, in the
presence of hapten, a strikingly higher percentage of DC died upon
coculture with T cells obtained from UV-tolerized mice (Table I, group c) as compared with those from
naive mice (group a). In contrast, the increase in DC death was much
less pronounced in cocultures with T cells obtained from sensitized
mice (group b). Enhanced DC death was not observed in the absence of
the specific hapten (group k) and when using allogeneic DC (group l).
Among T cells, activation-induced cell death was clearly evident in
primed (group b) as opposed to naive T cells (group a); however, no
additional increase was observed in T cells obtained from UV-tolerized
mice (group c). These data suggest that DC death during hapten
presentation is strongly increased in the presence of hapten-specific
suppressor cells.
|
|
T cells from UV-tolerized mice up-regulate Fas expression on DC
Since the coculture experiments revealed that T cells obtained
from UV-tolerized mice enhance DC death, and that this may be mediated
via the Fas/FasL system, we postulated that these T cells might induce
Fas expression on DC. To address this issue, DC obtained from C3H mice
were again cocultured in the presence of DNBS with T cells obtained
from naive, DNFB-sensitized or -tolerized C3H mice. Cells were stained
48 h later with an anti-Fas Ab, and FACS analysis was
performed. DC were gated, and Fas expression on DC was measured. DC
constitutively expressed Fas, and this expression was not affected by
the coincubation of DC with T cells from either naive or sensitized T
cells, while up-regulation of Fas expression was found upon
coincubation with T cells obtained from UV-tolerized mice (Fig. 4). Therefore, these data demonstrate
that T cells from UV-tolerized mice enhance Fas expression on
DC.
|
Recently, we and others reported that IL-12 can break established
UV-induced tolerance (16, 17, 18), although the underlying mechanism
remains unclear. Based on the data obtained from the coincubation
experiments, we hypothesized that IL-12 could counteract the inhibitory
activity of suppressor cells by preventing suppressor cell-mediated DC
death. Therefore, T cells from UV-tolerized mice and DC were
coincubated in the presence of rIL-12. The addition of IL-12 reduced
the number of dead DC (Table I, d). This suggests that IL-12 may break
tolerance by preventing DC from undergoing cell death induced by T
suppressor cells. Since the in vivo data indicated a crucial role of
the Fas/FasL system in the mediation of tolerance, we wondered whether
IL-12 could rescue DC from FasL-induced killing. Therefore, the DC line
XS52 was incubated with rFasL, and apoptosis was measured 16 h
later using a cell death ELISA. Treatment of XS52 cells with rFasL
resulted in pronounced cell killing (Fig. 5). In contrast, when XS52 cells were
preincubated with rIL-12 for 8 h before the addition of rFasL,
fewer cells died.
|
Since T suppressor cells induced Fas expression on DC, and since
IL-12 prevented DC from T suppressor cell-induced killing, we
postulated that IL-12 might rescue DC by down-regulating Fas expression
on DC. This appeared to be the case, as the addition of IL-12 to DC
coincubated with T cells from tolerized mice resulted in
down-regulation of Fas expression on DC (Fig. 4, bottom
panel). To further confirm this effect of IL-12, XS52 cells
were cultured in the presence of IL-12 or left untreated and evaluated
for Fas expression. FACS analysis revealed constitutive Fas expression
that could be additionally enhanced by IFN-. Preincubation with
IL-12 significantly down-regulated both constitutive and
IFN--induced Fas expression on XS52 cells (Fig. 6).
|
for the next 2 h. For control purposes, XS52
cells were either exposed to IFN- or IL-12 or left untreated. RNA
was extracted, and Northern blot was analysis performed using a cDNA
probe encoding murine Fas. IFN- significantly induced Fas mRNA
expression, which was reduced upon treatment with IL-12 (Fig. 7).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Since UV-induced tolerance is mediated by transferable hapten-specific
T suppressor cells (6), we also investigated whether suppressor cells
had developed in Fas- and FasL-deficient mice, respectively. Whereas
transfer of lymphocytes from UV-irradiated lpr or
gld mice into unirradiated, naive, syngeneic recipients
failed to inhibit subsequent sensitization, transfer of these cells
into unirradiated, naive C3H mice caused immune tolerance in the
recipients. Likewise, transfer of lymphocytes from UV-irradiated C3H
mice into naive, unirradiated lpr or gld mice
failed to induce hapten-specific tolerance (Fig. 2). Thus, 1)
suppressor cells are generated in Fas- and FasL-deficient mice; and 2)
to exert their immunomodulatory function, the expression of Fas and
FasL is required on the cells of the recipient but not on the
suppressor cells themselves. In a different experimental system,
Griffith et al. (14) recently showed that tolerance is induced via
Fas/FasL-dependent apoptosis of lymphocytes upon Ag injection into the
anterior chamber of the eye. The study demonstrated that Fas expression
on lymphoid cells and FasL expression on eye cells is required for the
induction of tolerance. In contrast, our data show that in the UV
model, the Fas/FasL system is not necessary for the induction of
tolerance-mediating suppressor cells, since these cells, obtained from
either lpr or gld mice, are functionally active
provided they are injected into a Fas/FasL-expressing donor. This
observation implies that T suppressor cells need the Fas/FasL system to
exert their inhibitory activity. However, since neither Fas nor FasL
need to be expressed on the suppressor cell for mediating tolerance
(Fig. 2, B and D), T suppressor cells
apparently do not induce apoptosis directly via Fas/FasL interaction
but rather via an indirect mechanism, e.g., by up-regulating Fas and
FasL expression on cells in the recipient.
This view is supported by the cross-transfer experiments shown in
Figure 2, which suggest that UV-induced T suppressor cells do not kill
their targets via the expression of FasL, as suppression is still
observed upon transfer of suppressor cells obtained from gld
mice which lack FasL. These data imply that suppressor cells induce
other cells in the recipient to undergo cell death via the Fas/FasL
pathway. Since APCs and hapten-specific T cells are critical during
sensitization, these cells would be potential targets. We assumed that
Th cells are a less likely candidate, since Glass et al. (24) observed
that suppressor cells fail to impair the CHS response of cotransferred
immune lymph node cells, indicating that hapten-specific T suppressor
cells do not exhibit the capacity to suppress effector cells which are
already primed against the relevant hapten. Thus, we hypothesized that
APCs may undergo cell death when presenting the hapten in the presence
of T suppressor cells.
To address this issue, T cells were isolated from C3H mice 5 days after sensitization through UV-exposed skin. Since suppression can be transferred at this time, T suppressor cells must already be present in these T cell suspensions. Since UV-induced T suppressor cells have not been phenotyped, and specific markers do not exist, it is not possible to isolate and purify the hapten-specific T suppressor cells. Therefore, we had to use bulk T cell suspensions obtained from UV-tolerized mice (i.e., hapten-treated through UV-exposed skin), and thus the term "T suppressor cells" refers to such bulk T cell suspensions. To investigate whether APCs undergo cell death in the presence of these T suppressor cells, DC were obtained from BM of C3H mice, coincubated with the T cell suspensions in the presence of the water soluble DNFB analogue, DNBS, and analyzed by flow cytometry. As this study revealed, a strikingly higher percentage of DC died upon coculture with T cells obtained from UV-tolerized mice as compared with those from naive or sensitized mice. This event is hapten-specific and MHC-dependent, because it is not observed in the absence of hapten or when DC obtained from allogeneic BALB/c mice were used. DC death appears to be due to apoptosis, because dead DC stained positively for annexin V (data not shown), a specific surface marker for apoptosis (25).
In analogy to our in vivo experiments, which indicated that the
transfer of tolerance is dependent upon the Fas/FasL system, DC from
lpr mice were much less sensitive to cell death in the
presence of T cells from UV-tolerized mice. Similar findings were
observed when using DC from gld mice, implying that, to
undergo suppressor cell-induced death, both Fas and FasL apparently
need to be expressed on DC. Although in many systems the death of Fas-
expressing cells is induced by other cells carrying FasL, T cells have
been shown to undergo programmed cell death due to Fas/FasL interaction
in a cell-autonomous manner (26, 27). Similarly, in Hashimoto’s
thyroiditis, thyrocytes undergo programmed cell death due to
cell-autonomous coexpression of Fas/FasL (28). Because neither Fas nor
FasL is required on the suppressor cells (as demonstrated by the
adoptive transfer experiments shown in Fig. 2), it is likely that the
two molecules become coexpressed on DC, thereby inducing an autocrine
suicide. This possibility is supported by previous reports indicating
that DC can express CD95 and CD95L (29, 30, 31). Accordingly, FACS analysis
revealed that Fas expression on DC is induced in the presence of T
suppressor cells, whereas no major differences were observed in FasL
expression (data not shown). However, FasL data must be interpreted
with caution, since the evaluation of FasL expression by FACS analysis
can be problematic due to the rapid shedding of FasL (32). While DC
obtained from gld or lpr mice were clearly
resistant to T suppressor cell-induced apoptosis, DC death was observed
when DC from gld and lpr mice were mixed. This
observation implies that not only autocrine but also paracrine suicide
may be relevant. On the other hand, it is important to mention that
changes in the death rates were observed not only for DC but also for T
cells. In particular, T cells obtained from either sensitized or
tolerized C3H mice died to a greater extent upon coincubation with DC
in the presence of the specific hapten. Although we did not pursue this
phenomenon, we interpreted it to be compatible with activation-induced
cell death (7). However, when similar experiments were performed with
DC from either gld or lpr mice, reduction of the
T cell death rate was observed, as was indeed unexpected. While reduced
T cell death upon coincubation with DC from gld mice is
explainable by the fact that the FasL expressed on DC is involved in
activation-induced T cell death, we do not as of yet have an
explanation as to why the same resistance of T cells was observed when
DC from lpr mice were used.
We are aware that the in vitro system we used is in some ways artificial and has a variety of limitations: 1) we cannot formally exclude that the suppressor cells may induce FasL expression of a third-party cell type, representing a minor contaminant in the DC preparation; 2) the major disadvantage is the use of bulk T cell suspensions as a source for T suppressor cells; and 3) although DC death was strikingly enhanced upon coculture with T cells from tolerized mice in the presence of the hapten, it is important to note that a certain degree of DC death was also observed in the control coculture experiments. In particular it appears that untolerized, Ag-specific T cells can also induce DC death to some extent, implying more of a quantitative and rather than a qualitative difference between suppressor and activating T cells. Nevertheless, despite these disadvantages, these data provide evidence as to how UV-induced T suppressor cells might act and also give an explanation for our in vivo data, which clearly show that the apoptosis-related Fas/FasL system is essential for UV-induced tolerance.
To further pursue this issue, we studied the effect of IL-12 on DC
death in the coculture experiments. Recently, we and others have
reported that IL-12 is able to prevent UV-induced inhibition of the
induction of CHS when injected i.p. between UV-exposure and hapten
sensitization (16, 17, 18). More importantly, it was observed that the
injection of IL-12 at a later time point, when T suppressor cells have
already developed, enables sensitization against the specific hapten.
However, the mechanism by which IL-12 breaks established tolerance is
still unclear. Due to its ability to counteract the activity of
suppressor cells, we were interested in determining whether IL-12
interferes with T suppressor cell-induced DC death. Indeed, the
addition of IL-12 to cocultures of DC and T suppressor cells
significantly reduced the number of dead DC. Since experiments using DC
from lpr and gld mice, respectively, indicated
that Fas/FasL expression appears essential for the induction of DC
death, we also asked whether IL-12 could interfere with Fas-mediated
apoptosis of DC. For this purpose, the DC line XS52 was treated with
rFasL, and the induction of apoptosis was measured. While rFasL induced
apoptosis of XS52 cells, preincubation of XS52 with rIL-12 before the
addition of rFasL significantly reduced the rate of apoptosis.
Moreover, T suppressor cells induced Fas surface expression on DC (Fig. 4), whereas IL-12 down-regulated Fas expression on DC. Since Northern
blot analysis revealed reduced levels of Fas-specific transcripts upon
IL-12 treatment, the inhibitory effect may be regulated at the mRNA
level. Although we regard down-regulation of Fas expression in DC as an
important event, by which IL-12 may protect DC from Fas-mediated cell
death, other mechanisms may also be involved, since IL-12 treatment
caused a reduction but not a complete suppression of Fas expression on
DC. In this respect, the induction of antiapoptotic genes such as
bcl-2, bcl-xL,
FLICE-inhibitory protein, Fas-associated phosphatase-1 (31, 33, 34, 35, 36)
must be considered as an additional possibility.
Together, these data provide evidence for an important link between the apoptosis-associated Fas/FasL system and immune tolerance by demonstrating that the Fas/FasL system is crucially involved in mediating UV-induced tolerance. The data further indicate that T suppressor cells can exert their suppressive activity in vivo only in the presence of a functionally active Fas/FasL system. Based on our in vitro data, we speculate that UV-induced T suppressor cells may cause the cell death of APCs in the presence of the specific hapten via the Fas/FasL system. IL-12 can rescue APCs from suppressor cell-induced cell death, a mechanism possibly responsible for the unique ability of IL-12 to break established immune tolerance.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Thomas Schwarz, Department of Dermatology, University Münster, Von-Esmarchstrasse 56, D-48149 Münster, Germany. E-mail address:
3 Abbreviations used in this paper: CHS, contact hypersensitivity; DC, dendritic cells; BM, bone marrow, DNFB, 2,4-dinitrofluorobenzene; DNBS, 2,4-dinitrobenzenesulfonic sodium salt; FasL, Fas ligand; FSC, forward light scatter; PI, propidium iodide; SSC, side light scatter.
Received for publication July 28, 1997. Accepted for publication January 5, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. Maeda, A. Schwarz, A. Bullinger, A. Morita, D. Peritt, and T. Schwarz Experimental Extracorporeal Photopheresis Inhibits the Sensitization and Effector Phases of Contact Hypersensitivity via Two Mechanisms: Generation of IL-10 and Induction of Regulatory T Cells J. Immunol., November 1, 2008; 181(9): 5956 - 5962. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Pradhan, H. K. Kim, C. J. Thrash, M. A. Cox, S. K. Mantena, J.-H. Wu, M. Athar, S. K. Katiyar, C. A. Elmets, and L. Timares A Critical Role for the Proapoptotic Protein Bid in Ultraviolet-Induced Immune Suppression and Cutaneous Apoptosis J. Immunol., September 1, 2008; 181(5): 3077 - 3088. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Maeda, S. Beissert, T. Schwarz, and A. Schwarz Phenotypic and Functional Characterization of Ultraviolet Radiation-Induced Regulatory T Cells J. Immunol., March 1, 2008; 180(5): 3065 - 3071. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Schwarz, A. Maeda, and T. Schwarz Alteration of the Migratory Behavior of UV-Induced Regulatory T Cells by Tissue-Specific Dendritic Cells J. Immunol., January 15, 2007; 178(2): 877 - 886. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nowak, F. Kopp, K. Roelofs-Haarhuis, X. Wu, and E. Gleichmann Oral nickel tolerance: fas ligand-expressing invariant NK T cells promote tolerance induction by eliciting apoptotic death of antigen-carrying, effete B cells. J. Immunol., April 15, 2006; 176(8): 4581 - 4589. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Riemann, K. Loser, S. Beissert, M. Fujita, A. Schwarz, T. Schwarz, and S. Grabbe IL-12 Breaks Dinitrothiocyanobenzene (DNTB)-Mediated Tolerance and Converts the Tolerogen DNTB into an Immunogen J. Immunol., November 1, 2005; 175(9): 5866 - 5874. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Barreiro, G. Luker, J. Herndon, and T. A. Ferguson Termination of Antigen-Specific Immunity by CD95 Ligand (Fas Ligand) and IL-10 J. Immunol., August 1, 2004; 173(3): 1519 - 1525. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Gorbachev and R. L. Fairchild CD4+ T Cells Regulate CD8+ T Cell-Mediated Cutaneous Immune Responses by Restricting Effector T Cell Development through a Fas Ligand-Dependent Mechanism J. Immunol., February 15, 2004; 172(4): 2286 - 2295. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Schwarz, A. Maeda, M. K. Wild, K. Kernebeck, N. Gross, Y. Aragane, S. Beissert, D. Vestweber, and T. Schwarz Ultraviolet Radiation-Induced Regulatory T Cells Not Only Inhibit the Induction but Can Suppress the Effector Phase of Contact Hypersensitivity J. Immunol., January 15, 2004; 172(2): 1036 - 1043. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Aragane, A. Maeda, A. Schwarz, T. Tezuka, K. Ariizumi, and T. Schwarz Involvement of Dectin-2 in Ultraviolet Radiation-Induced Tolerance J. Immunol., October 1, 2003; 171(7): 3801 - 3807. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Wachter, M. Averbeck, H. Hara, J. P. Tesmann, J. C. Simon, C. C. Termeer, and R. W. Denfeld Induction of CD4+ T Cell Apoptosis as a Consequence of Impaired Cytoskeletal Rearrangement in UVB-Irradiated Dendritic Cells J. Immunol., July 15, 2003; 171(2): 776 - 782. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hoves, S. W. Krause, D. Halbritter, H.-G. Zhang, J. D. Mountz, J. Scholmerich, and M. Fleck Mature But Not Immature Fas Ligand (CD95L)-Transduced Human Monocyte-Derived Dendritic Cells Are Protected from Fas-Mediated Apoptosis and Can Be Used as Killer APC J. Immunol., June 1, 2003; 170(11): 5406 - 5413. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Wirleitner, D. Reider, S. Ebner, G. Bock, B. Widner, M. Jaeger, H. Schennach, N. Romani, and D. Fuchs Monocyte-derived dendritic cells release neopterin J. Leukoc. Biol., December 1, 2002; 72(6): 1148 - 1153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Ferguson, J. Herndon, B. Elzey, T. S. Griffith, S. Schoenberger, and D. R. Green Uptake of Apoptotic Antigen-Coupled Cells by Lymphoid Dendritic Cells and Cross-Priming of CD8+ T Cells Produce Active Immune Unresponsiveness J. Immunol., June 1, 2002; 168(11): 5589 - 5595. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Frasca, C. Scotta, G. Lombardi, and E. Piccolella Human Anergic CD4+ T Cells Can Act as Suppressor Cells by Affecting Autologous Dendritic Cell Conditioning and Survival J. Immunol., February 1, 2002; 168(3): 1060 - 1068. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Walterscheid, S. E. Ullrich, and D. X. Nghiem Platelet-activating Factor, a Molecular Sensor for Cellular Damage, Activates Systemic Immune Suppression J. Exp. Med., January 14, 2002; 195(2): 171 - 179. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Schwarz, S. Beissert, K. Grosse-Heitmeyer, M. Gunzer, J. A. Bluestone, S. Grabbe, and T. Schwarz Evidence for Functional Relevance of CTLA-4 in Ultraviolet-Radiation-Induced Tolerance J. Immunol., August 15, 2000; 165(4): 1824 - 1831. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Pirtskhalaishvili, G. V. Shurin, A. Gambotto, C. Esche, M. Wahl, Z. R. Yurkovetsky, P. D. Robbins, and M. R. Shurin Transduction of Dendritic Cells with Bcl-xL Increases Their Resistance to Prostate Cancer-Induced Apoptosis and Antitumor Effect in Mice J. Immunol., August 15, 2000; 165(4): 1956 - 1964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Kiertscher, J. Luo, S. M. Dubinett, and M. D. Roth Tumors Promote Altered Maturation and Early Apoptosis of Monocyte-Derived Dendritic Cells J. Immunol., February 1, 2000; 164(3): 1269 - 1276. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nakagawa, C. W. Koomen, J. D. Bos, and M. B. M. Teunissen Differential Modulation of Human Epidermal Langerhans Cell Maturation by Ultraviolet B Radiation J. Immunol., November 15, 1999; 163(10): 5192 - 5200. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Hill, V. K. Shreedhar, M. L. Kripke, and L. B. Owen-Schaub A Critical Role for Fas Ligand in the Active Suppression of Systemic Immune Responses by Ultraviolet Radiation J. Exp. Med., April 19, 1999; 189(8): 1285 - 1294. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Kothny-Wilkes, D. Kulms, B. Poppelmann, T. A. Luger, M. Kubin, and T. Schwarz Interleukin-1 Protects Transformed Keratinocytes from Tumor Necrosis Factor-related Apoptosis-inducing Ligand J. Biol. Chem., October 30, 1998; 273(44): 29247 - 29253. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. Q. Vu, C. D. Bortner, and J. A. Cidlowski Differential Involvement of Initiator Caspases in Apoptotic Volume Decrease and Potassium Efflux during Fas- and UV-induced Cell Death J. Biol. Chem., September 28, 2001; 276(40): 37602 - 37611. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
