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Departments of
*
Ophthalmology and Visual Sciences and
Pathology, Washington University School of Medicine, St. Louis, MO 63110; and
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Fas/FasL system is not only critical for immune privilege
(3, 6, 7), but also is important in the development of the
immune response, homeostasis of the immune system, and maintenance of
self-tolerance (8, 9). Additionally, Fas-mediated
apoptosis is involved in T cell cytotoxicity, tumorigenesis, and liver
disease (10, 11, 12). Fas is a type I membrane protein
belonging to the TNFR superfamily, which includes the p55 (TNFR1) and
p75 (TNFR2) TNFRs, CD40, 4-1BB, and the family of TNF-related
apoptosis-inducing ligand receptors, among others (13, 14). Cross-linking of Fas by FasL or specific Ab initiates the
cell death cascade, with the binding of Fas-associated death domain
protein (FADD) to the cytoplasmic domain of Fas being the first
molecular event (15, 16). This facilitates the binding and activation
of caspase-8 (FADD-like IL-1
-converting enzyme), followed by the
activation of other downstream caspases, degradation of DNA, and
ultimately cell death (17, 18). This process is tightly
regulated and depends, in the case of T cells, on prior cellular
activation. Interestingly, elements of the immune response (such as
IL-2; Ref. 19) potentiate Fas-mediated death through the
modulation of the apoptosis regulatory protein FADD-like
IL-1-converting enzyme inhibitory protein (FLIP)
(20).
FLIP was originally described as a viral product (vFLIP) (21, 22), but has since been shown to exist in mammalian cells (cFLIP). cFLIP mRNA exists as multiple splice variants, but protein expression is limited to a long and short form (cFLIPL and cFLIPS) (21). cFLIPS contains two death effector domains (DED) and is structurally related to vFLIP, whereas cFLIPL contains an additional caspase-like domain lacking catalytic activity. The DED of both FLIPL and FLIPS enable them to bind to the DED of FADD and caspase-8, preventing their recruitment to the death domain of Fas. This prevents formation of the death-inducing signaling complex and inhibits apoptosis (23, 24). The resistance of naive T cells to Fas-mediated death has been attributed to high intracellular levels of FLIP, which decrease following cellular activation and IL-2 production, thereby making the T cells susceptible to Fas-mediated death (20, 23). In addition to protecting cells from Fas-induced apoptosis, FLIP can protect cells from apoptosis induced by TNF, NF receptor-related apoptosis-mediated protein, and TNF-related apoptosis-inducing ligand. FLIP has also been found in heart, skeletal muscle, and kidney (23) where it is presumed to function as an antiapoptotic factor to help maintain organ homeostasis. Expression in tumors promotes tumor growth presumably by blocking the cell death pathway (25, 26). The importance of FLIP was recently demonstrated by the embryonic lethality of targeted deletion of this molecule (27).
In our studies of FasL-induced death in the eye, we have observed that apoptotic cell death occurs rapidly in both activated and freshly isolated lymphoid cells (3, 4). Since Fas-mediated apoptosis is tightly regulated, we suspected that other factors might contribute to this process. In the present study, we examined the role of the proinflammatory cytokine TNF in the regulation of Fas-mediated apoptosis in the eye. Our results show that TNF, via binding to TNFR2, increases the sensitivity of T cells to Fas-mediated death by modulating the expression of a molecule that regulates apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c and C57BL/6 were purchased from the National Cancer Institute. TNFR1-/-, TNFR2-/-, and normal littermate mice were purchased from The Jackson Laboratory (Bar Harbor, ME). In all in vivo experiments, groups consisted of five or more animals and were used at 6–10 wk of age. Experiments were repeated at least three times with similar results.
Reagents
2,4,6-Trinitro-1-chlorobenzene (TNCB) was purchased from Eastern Chemical (Smithtown, NY). 2,4,6-Trinitrobenzenesulfonic acid (TNBS) was purchased from Sigma (St. Louis, MO). Hamster Ab to murine TNF (TN3.19.12) was provided by Dr. R. Schreiber (Washington University, St. Louis, MO). Recombinant murine TNF was purchased from Genzyme (Cambridge, MA). Jo2 (anti-Fas/CD95), anti-CD3-FITC, and streptavidin-PE were obtained from BD PharMingen (San Diego, CA). Goat anti-TNFR Abs (TNFR1 and TNFR2) and TUNEL stain kit for flow cytometry (catalogue no. TA5354) were purchased from R&D Systems (Minneapolis, MN). Anti-goat conjugated with FITC or PE was purchased from Jackson ImmunoResearch (Broomall, PA). The anti-FLAG M2 mAb for aggregating FLAG-tagged soluble FasL (sFasL) (28) was purchased from Eastman Kodak (Rochester, NY).
2,4,6-Trinitrophenyl (TNP) coupling of T cells
T cells were isolated from spleen cell suspensions and coupled with TNP as previously described (29, 30). TNP-T cells were used for all anterior chamber (AC) injections, while TNP-spleen cells were used in the RT-PCR analysis for FLIP.
AC inoculations
Mice were lightly anesthetized with Metofane (methoxyflurane) and injections were done under a dissecting microscope. Each intracameral injection contained 5 x 105 TNP-T cells or 2.5 x 104 PFU HSV-1 (KOS) in a 0.005-ml volume using a 0.25-ml Hamilton Microliter syringe (Hamilton, Reno, NV) fitted with a 33-gauge needle.
Tissue extraction and TNF ELISA
At various times following AC injection of TNP-coupled spleen cells, eyes were removed and added to a 1.5-ml microcentrifuge tube. To each sample, 0.5 ml of 0.1% Tween 20 in PBS (PBS/Tween) was added and the samples were ground using a plastic pellet pestle. Samples were quick frozen in liquid N2, thawed in a 37°C water bath, and ground again. Samples were then centrifuged for 5 min at 13,000 x g. Supernatants were removed for TNF determination.
TNF protein levels were quantitated using a sandwich ELISA developed with two anti-TNF mAb (31). Ninety-six-well ELISA plates (Immulon II; Dynatech Laboratories, Chantilly, VA) were coated with 3.5 µg/ml purified TN3 19.12 mAb (30) diluted in coating buffer (0.1 M NaHCO3/0.1 M Na2CO3) for 2 h at 37°C. Wells were blocked with DMEM-10% FCS for 30 min at room temperature. Plates were washed three times with PBS/Tween and the TNF standard or test samples were added (100 µl/well). Plates were incubated overnight at 4°C, wells were washed four times with PBS/Tween, and 100 µl/well polyvalent goat anti-recombinant murine TNF (diluted 1:500 in DMEM-10% FCS; provided by Dr. K. Sheehan, Washington University) was added for 1 h at room temperature. Wells were washed four times, followed by the addition of 100 µl/well peroxidase-conjugated rabbit anti-goat IgG (diluted 1/2000 in DMEM-10% FCS; ICN Pharmaceuticals, Costa Mesa, CA) for 1 h at room temperature. Wells were washed four times with PBS/Tween and 100 µl/well ABTS (0.1 M sodium citrate, 1 mM ABTS, 0.016% (v/v) H2O2) was added. Upon color development, the plate was analyzed on an ELISA plate reader at A414.
In situ TUNEL staining
At various times following AC injection of TNP-coupled cells, eyes were removed, fixed in Formalin, and processed for paraffin sectioning. Ten-micrometer sections were mounted onto microscope slides and incubated overnight at 55°C. Sections were then deparaffinized by washing for 5 min in xylene twice, 5 min in absolute ethanol twice, 3 min in 95% ethanol, 3 min in 70% ethanol, and 5 min in PBS. Protein present in the sections was digested with 20 µg/ml proteinase K for 20 min at room temperature. Following four washes in distilled water, endogenous peroxidase was quenched with 2.0% H2O2 for 5 min at room temperature and sections were washed twice in PBS. Labeling of 3'-OH fragmented DNA ends was performed with an in situ apoptosis detection kit (ApopTag; Oncor, Gaithersburg, MD) following package instructions. Detection of labeled ends was done with the kit-supplied anti-digoxigenin-peroxidase Ab and development of diaminobenzidine substrate (Vector Laboratories, Burlingame, CA).
RT-PCR
Total RNA was isolated with TRIzol reagent (Life Technologies,
Gaithersburg, MD) as per the manufacturer’s instructions. RNA samples
(1 µg each) were tested for DNA contamination by 30 cycles of PCR
with mouse -actin primers. After it was shown there was no DNA
contamination, cDNA synthesis was performed using an RNA PCR kit
(PerkinElmer, Norwalk, CT) with the supplied
oligo(dT)16 primer. Reverse transcription was
performed using a thermal program of 25°C for 10 min, 42°C for 30
min, and 95°C for 5 min. PCR were performed using the following
primers: -actin (forward, 5'-TGGAATCCTGTGGCATCCATGAAAC-3'; reverse,
5'-TAAAACGCAGCTCAGTAACAGTCCG-3'); and FLIP (forward,
5'-CAAGATAGCCAAGGACAAGAG-3'; reverse, 5'-GATGGATGTCTTCACCAGTG-3');
Bax (forward, 5'-TTTATGGAGGGGTCCGGGGA-3'; reverse,
5'-TGTCCAGCCCATGATGGTTCT-3') giving products of 349, 307, and 420 bp,
respectively. -Actin PCR cycle conditions were 95°C for 1 min,
55°C for 1.5 min, and 72°C for 2 min for 22 cycles. FLIP PCR cycle
conditions were 95°C for 1 min, 61°C for 1 min, and 72°C for 1.5
min for 35 cycles. Bax PCR cycle conditions were 94°C for 2 min,
65°C for 1.5 min, and 72°C for 1.5 min for 35 cycles. Samples were
resolved on a 2% agarose gel and visualized with ethidium bromide.
Western blot for FLIP
Cellular proteins from 4 x 105 cells were separated by SDS-PAGE on a 10% polyacrylamide gel under reducing conditions and then transferred to nitrocellulose membranes by semidry electroblotting. Ponceau S (Sigma) staining of the nitrocellulose confirmed the equal loading of proteins. Membranes were blocked overnight with 5% nonfat dry milk in PBS and incubated with anti-FLIP (AL 109) (20, 23) at a dilution of 1/400 for 1 h. Membranes were washed three times and then incubated with peroxidase-conjugated goat anti-rat IgG (Amersham, Arlington Heights, IL) for 1 h. The membranes were washed thoroughly with PBS/0.05% Tween 20 and developed by chemiluminescence according to the manufacturer’s protocol (ECL; Amersham Pharmacia Biotech, Piscataway, NJ).
Induction of immune deviation
Immune deviation was induced as described elsewhere (29). Briefly, TNP-coupled spleen cells were injected into the AC, and then the mice were immunized 48 h after AC injection with 0.050 ml of 1% TNCB in acetone/olive oil (3:1) applied to shaved abdominal skin. Five days later, mice were challenged with 0.033 ml of 10 mM TNBS in PBS in the right footpad and 0.033 ml of PBS in the left footpad. Values are expressed in micrometers (±SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge). Background values represent the difference between the challenged and unchallenged foot in unimmunized mice.
Isolation of ocular inflammatory cells
Twenty-four or 48 h following AC injection of HSV-1, mice were enucleated and eyes were minced between the ends of two frosted glass slides in HBSS without Ca2+/Mg2+ containing 10 mM EDTA. Large pieces were removed by filtration through 70-µm cell strainers and mononuclear cells were washed three times with HBSS. Following wash, cells were counted and yields were typically 1 x 106/injected eye. Cells were then analyzed by flow cytometry.
Flow cytometry (FACS)
FACS staining was performed by standard protocols. Briefly, for surface labeling, 1 x 105 spleen cells/ml were incubated with 20 µl of 10 µg/ml goat anti-TNFR1 or TNFR2 for 30 min at 4°C. Cells were washed twice in staining buffer (2% FCS in PBS plus 0.01% NaN3) and resuspended in 20 µl of donkey anti-goat FITC (15 µg/ml) for 30 min at 4°C. Cells were then washed twice with staining buffer, resuspended in 400 µl of 2% formaldehyde, and collected on a BD Biosciences FACSCalibur (Mountain View, CA). In some cases, cells were also stained with anti-CD3-PE and analyzed for CD3 and TNFR expression. To measure TUNEL-positive lymphocytes in the eye, cells were isolated and surface stained as described above. Cells were then fixed, permeabilized, incubated with TdT and biotinylated dNTP per kit instructions (R&D Systems) and stained with streptavidin-PE. All events were analyzed using CellQuest software.
Cell death assay (DO11.10 T cell hybridoma)
DO11.10 cells (4 x 105/ml) were labeled for 2 h at 37°C with 5 µCi/ml [3H]thymidine. Following three washes in culture medium, cells (2 x 104/well of a 96-well plate) were incubated with medium alone, murine TNF, Jo2, or TNF plus Jo2 for 16 h at 37°C. Unfragmented DNA was collected by filtration through glass fiber filters (Packard Instrument, Meriden, CT) using a Filtermate 96 cell harvester (Packard Instrument) and counted on a Microplate scintillation counter (Packard Instrument). Percent DNA fragmentation was determined by the formula (cpm without RPE cells - cpm with RPE cells) divided by the cpm without RPE cells (x100). Treatments were done in triplicate.
Cell death assays (spleen cells)
Spleen cells (1 x 106/ml) were cultured with TNF or goat anti-mouse TNFR (R1 or R2) for 6 h at 37°C in 1 ml of RPMI 1640 with 0.5% normal mouse serum. Cells were collected, concentrated by centrifugation, and added to 96-well plates (Immunlon II; Dynatech Laboratories) containing immobilized Jo2. Cells were incubated for an additional 16 h and cell death was determined by trypan blue exclusion. Immobilized Jo2 was prepared by first coating plates with 5 µg/ml rabbit anti-goat Ab for 2 h at 37°C. Following three washes with PBS, 5 µg/ml Jo2 was added for 2 h at 37°C. Cells were added following a three times wash with PBS. Death was assessed by trypan blue exclusion.
For T cell killing by sFasL and TNF, CD4+ T cells were obtained from CD8-/- spleen cells by purification over antimouse Ig columns. Typical yields were 85% CD4+ T cells (data not shown). Cells were placed in 96-well flat-bottom culture plates in triplicate wells (2 x 105/well) in 100 µl of complete RPMI 1640 (10% FCS, 2 mM L-glutamine, 0.05 mM 2-ME, 10 mM HEPES, 1 mM sodium pyruvate, and antibiotics) and incubated overnight with 100 ng/ml murine TNF in a 5% CO2 humidified 37°C incubator. Wells were then given FLAG-tagged sFasL (100 ng/ml) for 1/2 h before adding aggregating anti-FLAG Ab (M2, 2 µg/ml) and incubating for 24 h at 37°C. To measure apoptotic cell death, subdiploid DNA analysis was performed. Cells were collected and washed in buffer (2% FCS in PBS plus 0.01% NaN3) and fixed at room temperature for 30 min in EtOH. Cells were then resuspended in 500 µl of buffer plus 50 µg/ml propidium iodide. Apoptosis was determined by gating on the population containing subdiploid DNA using CellQuest software after data acquisition on a BD Biosciences FACSCalibur.
Statistical analysis
Significant differences between groups were evaluated using a two-tailed Student’s t test (p < 0.01).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies from our laboratory have shown activated and freshly isolated lymphoid cells entering the AC of the eye rapidly undergo apoptotic cell death (3, 4). This death is induced by the FasL expression on ocular tissues and is a prerequisite for the induction of immune deviation to the Ags encountered in the AC (4, 5). The production of TNF is also an important event in this phenomenon since TNF mRNA levels peak shortly after AC injection and the coinjection of a neutralizing mAb to TNF with the Ag blocked immune deviation (32). Since members of the TNF family cooperate in the induction of apoptosis (33, 34), we tested for a link between TNF production and FasL-induced death.
Following AC injection of TNP-T cells, TNF protein levels increased
rapidly over the first 2–3 h before falling to near normal levels by
9 h (Fig. 1
). In situ TUNEL staining
24 h following AC injection showed numerous apoptotic cells (Fig. 2A), confirming previous
observations (4). However, when a neutralizing
anti-TNF mAb was coinjected with the TNP-T cells, apoptosis was
prevented (Fig. 2B). The role of TNF in cell death was then
examined using TNP-T cells from TNFR1-/- or
TNFR2-/- mice. Although
TNFR1-/- TNP-T cells underwent apoptosis in the
AC much like the normal TNP-T cells, TNFR2-/-
TNP-T cells did not (Fig. 2, C and D,
respectively).
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D), a prediction is that immune
deviation cannot be induced with these cells. To test this, we compared
the ability of normal, TNFR1-/- and
TNFR2-/- TNP-T cells to induce immune deviation
in normal B6 mice. Upon AC injection, we found that TNP-T cells from
the normal and TNFR1-/- mice induced immune
deviation, whereas those from TNFR2-/- or
TNFR1/2-/- mice did not (Fig. 3).
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A). Therefore, the
apparent preference for TNFR2 may be because TNFR1 is simply absent
from the lymphoid cell population.
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, B and C, show
that wild-type cells entering the eye in response to HSV-1 express both
TNFR1 and TNFR2 at significant levels. We then examined apoptosis in
TNFR1-/- and TNFR2-/-
mice and found that only cells invading the
TNFR1-/- eye were TUNEL+,
whereas there was little or no apoptosis in the eyes of
TNFR2-/- mice (Fig. 5). Thus, FasL-induced cell death is
facilitated only in the presence of an active TNFR2. Collectively,
results in
Figs. 1–5 suggest that TNF signaling through TNFR2 is an
integral part of FasL-induced apoptosis of cells in the eye. When cells
lack TNFR2, they do not undergo apoptosis and they do not induce immune
deviation because they fail to receive a "sensitizing" signal
from TNF.
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To further explore the role of TNF in regulating Fas-mediated
apoptosis, we tested whether TNF could alter the sensitivity of cells
in vitro to Fas-mediated death. When T cell hybridoma DO11.10 cells
(high expressers of both TNFRs by FACS analysis; data not shown) were
cultured with TNF, minimal apoptosis was observed (Fig. 6A). Similarly, culturing the
cells with anti-Fas Ab (Jo2) resulted in a modest level of killing
only at the highest concentration tested. However, when noncytotoxic
concentrations of TNF and anti-Fas were used together, a
substantial increase in cell death was observed. This suggests that TNF
and Fas can cooperate to induce cell death in this hybridoma.
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extend these
observations to splenic cells using TNF or agonistic Abs to the TNFRs.
In Fig. 7A, only the combination of TNF and anti-Fas
resulted in measurable cell death, while data in Fig. 7B
show that inclusion of anti-TNFR2 (but not anti-TNFR1) at 0.1
and 1.0 µg/ml sensitized cells for death on anti-Fas Ab. Although
1.0 µg/ml anti-TNFR1 with anti-Fas appears to protect cells
from death, the data did not reach statistical significance. Data in
Table I extend these observations to the
natural ligand for Fas and show that only in the presence of TNF and
sFasL do CD4+ T cells undergo significant
apoptosis as measured by subdiploid DNA analysis. We conclude from
these data that FasL-induced apoptosis of lymphoid cells in the eye is
assisted by TNF via the TNFR2 receptor.
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Next, we explored the potential mechanisms by which TNF signaling through TNFR2 could facilitate apoptosis in lymphoid cells via Fas. Possible mechanisms for this effect could be increased Fas expression, increased expression of pro-apoptotic molecules, and/or down regulation of antiapoptotic molecules. When we examined Fas expression, we observed that TNF did not significantly increase the cell surface expression of Fas measured by flow cytometry (data not shown).
We then considered molecules that regulate apoptosis. Bcl-2 family
members are known to regulate cell death by repressing (i.e., Bcl-2,
Bcl-xL, Mcl-1, and A1) or promoting (i.e., Bax,
Bcl-xS, Bad, and Bak) the apoptotic process
(36). These molecules form homodimers and heterodimers
that are key to the induction of apoptosis (37, 38).
Another potent regulator of cell death is FLIP, which is a crucial
repressor of apoptosis (23). Fig. 6B shows that
incubation of DO11.10 hybridoma T cells with TNF significantly
decreased the levels of FLIP. In addition, mRNA levels for the
pro-apoptotic molecule Bax were elevated following treatment of cells
with TNF (Fig. 6C) (mRNA levels for Bcl-2 and
Bcl-xL were unchanged; data not shown).
Treatment of normal spleen cells with TNF or agonistic anti-TNFR
Abs also revealed modulation of the expression of Bax and FLIP.
Treatment of B6 spleen cells with anti-TNFR2 increased the level of
Bax in these cells (Fig. 8A).
Whereas FLIP transcripts were detectable in untreated B6,
TNFR1-/-, and TNFR2-/-
spleen cells, incubation with TNF resulted in the disappearance of FLIP
mRNA only in B6 or TNFR1-/- spleen cells (Fig. 8B). FLIP mRNA was still detectable in the
TNFR2-/- spleen cells after incubation with
TNF, further supporting the hypothesis that signaling through TNFR2 is
necessary for sensitization to Fas-mediated apoptosis. (Note: levels of
Bcl-2 or Bcl-xL mRNA remained unaltered after
incubation with agonistic TNF or anti-TNFR Abs (data not
shown).)
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies from our laboratory have revealed that TNF mRNA levels rapidly increase over the first 2–3 h after AC injection of TNP-spleen (32). In the present studies, TNF protein levels were found to follow similar kinetics after AC injection, with intraocular levels peaking 2 h after injection before returning to background levels by 8–9 h. These kinetics were similar to those found in the skin during the initiation of a contact sensitivity reaction, where TNF can be detected within 0.5–1 h following Ag application (39). It is interesting that TNF protein levels increase almost immediately after cell injection into the AC. We have previously observed that TNF mRNA production was the result of the interaction of T cells with ocular tissue (32). Thus, a possible scenario might be that ocular cells produce TNF in response to lymphoid cell invasion leading to increased sensitivity of the lymphocytes to killing by constitutive FasL. Another possibility is that the lymphoid cells make TNF in response to other elements in the ocular milieu, leading to their destruction by FasL. A third situation would be relevant to inflammatory reactions in the eye where lymphoid cells entering the eye in response to infection produce TNF. Under normal circumstances, this cytokine would promote the immune reaction (39); however, in the immune privileged site constitutive FasL rapidly kills TNF-sensitized lymphocytes before damage can be done.
In both splenic T cells and inflammatory cells responding to HSV-1, the
activity of TNF observed was mediated through the p75 (or TNFR2)
receptor. Although the exclusive expression of TNFR2 may explain the
results using splenic T cells, this explanation is not sufficient to
account for apoptosis of inflammatory cells responding to HSV-1. These
cells express both receptors but are killed by FasL following the
interaction of TNF with TNFR2. The dominance of TNFR2 may reflect the
higher affinity of TNFR2 for the ligand (40). This may
have relevance to immune privilege by allowing lower levels of TNF to
sensitize lymphoid cells for death in the eye. An effect mediated
through a high-affinity receptor would also provide a way to sensitize
cells that are early in activation with the relatively low levels of
TNF available in the eye (Fig. 1; Ref. 32). Thus, TNFR2
may have a specific role in pro-apoptotic and anti-inflammatory
responses leading to the protection of immune privileged sites.
The two TNF receptors are coexpressed in many tissues, including hemopoietic cells, although membrane expression is independently regulated and may differ depending on the cell type (41, 42, 43). It is now known that these receptors activate distinct signal pathways and can function independently (44, 45, 46). However, studies have shown that they can cooperate to induce cell death (34, 47), but the mechanism is unclear. It has been suggested that TNFRs cooperate at the level of receptor-ligand interactions (40) (e.g., the ligand-passing model); however, this does not account for the critical role of intracytoplasmic signaling in the function of both receptors. Recent studies have suggested that the TNFR-associated factor (TRAF) 1/TRAF-2 binding domain in TNFR2 cooperates with TNFR1 to induce apoptosis of PC60 and HeLa cells (34, 47). Although it is possible that TRAF2 could recruit inhibitory molecules such as FLIP away from Fas, our studies show that FLIP synthesis is inhibited by the TNF-TNFR2 interaction.
TNFR2 has been shown to induce the proliferation of submaximally activated naive cells, but apoptosis late in the activation process (48, 49). A recent report showed that CD8+ T cells are resistant to activation induced cell death (AICD) if they do not express TNFR2 (50). Although it has been reported that activated CD8+ T cells are most sensitive to apoptosis via the TNFRs (35, 50, 51), we have observed that nearly all mononuclear cells entering the eye, whether they are CD4+, CD8+, or non-T cells, are killed via the Fas/TNFR2 pathway. Similarly, freshly isolated splenic CD4+ and CD8+ T cells are killed in vitro by the interaction of Fas and TNFR2 (present studies, data not shown). To our knowledge, this is the first demonstration that cooperation between signals delivered to the cell through TNFR2 and Fas has relevance in vivo, i.e., to the maintenance of immune privilege and the induction of immune tolerance in the eye.
Our data show that cells infiltrating the AC at 24 or 48 h are susceptible to TNFR2/Fas-mediated death; however, their exact activation status is unknown. This may be an important consideration because it has been reported that T cells rapidly and transiently shed TNFRs during the first stage of Ag responsiveness (52). Our data do not address this; however, cells entering the eye in our model seem to be universally susceptible to death via Fas if they are expressing TNFR2.
Our results show that TNF affects several components of the cell death machinery. TNF-TNFR2 interaction alters the expression of the intracellular regulators of apoptosis, Bax and FLIP. Bax is a pro-apoptotic member of the Bcl-2 family of proteins that regulates cell death through the formation of homodimers and heterodimers with Bcl-2. It has been suggested that the ratio of Bcl-2:Bax determines cell survival or death following an apoptotic stimulus (36, 37, 38, 53). Following incubation with TNF, Bax mRNA levels rapidly increase, suggesting that the amount of Bcl-2 within the cell could no longer prevent Bax from forming homodimers and promoting cell death. We have previously demonstrated that Bcl-xL overexpression in T cells inhibits apoptosis of these cells in the eye (4). In this instance, excess Bcl-xL probably inhibited Bax homodimer formation and blocked cell death.
We also observed that TNF, acting through the TNFR2, caused a decrease in the intracellular levels of FLIP mRNA and protein. The expression of FLIP in T cells depends on the degree of cellular activation, with high levels present in naive T cells as well as T cells early in the activation process (23). FLIP levels decline with prolonged activation, in direct correlation with the T cell’s increased sensitivity to Fas-mediated apoptosis. FLIP has also been found to play a role in AICD of lymphocytes (20). AICD is an important mechanism of self-tolerance mediated by Fas-FasL interactions and enhanced by IL-2 (19). Interestingly, analysis of the biochemical mechanisms of IL-2-enhanced Fas-mediated T cell apoptosis revealed that IL-2 induces expression of FasL and simultaneously suppresses the transcription and expression of FLIP (20). Our observation that FLIP transcription decreased with TNF signaling through TNFR2 is similar to these observations with IL-2. Thus, these cytokines may play parallel roles in terminating an immune response. In the periphery, the growth-promoting cytokine IL-2 sensitizes T cells for death through suicide and fratricide. This permits an immune response but prevents excessive immune reactions that could lead to autoimmunity. In immune privileged sites, the proinflammatory cytokine TNF sensitizes cells entering the eye for death by constitutive FasL expression. This prevents the evolution of the immune response to protect the function of the visual axis.
When considering the mechanisms of immune privilege, it is important to
realize that multiple factors are responsible for this biological
phenomenon, including immunosuppressive cytokines (54, 55), neuropeptides (56), proinflammatory cytokines
(32), and FasL (3). We initially examined a
number of these factors (including TGF- and vasoactive intestinal
peptide) to determine their complicity with FasL for the
induction of cell death in the eye. These mediators had no effect on
the induction of Fas-mediated death in our system (data not shown). The
antiproliferative properties of these molecules (particularly TGF-)
are likely the underlying reason for their contribution to immune
privilege (54). The only factor we have found thus far
that promotes FasL-induced death in the eye is the proinflammatory
cytokine TNF, where it functions with FasL to protect the eye from the
damaging effects of immune and inflammatory reactions.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Urology, University of Iowa, Iowa City, IA 52242.
3 Address correspondence and reprint requests to Dr. Thomas A. Ferguson, Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, 660 South Euclid, Box 8096, St. Louis, MO 63110. E-mail address: Ferguson{at}vision.wustl.edu
4 Abbreviations used in this paper: FasL, Fas ligand; FADD, Fas-associated death domain protein; FLIP, FADD-like IL-1-converting enzyme inhibitory protein; AC, anterior chamber; AICD, activation-induced cell death; vFLIP, viral FLIP; cFLIP, cellular FLIP; DED, death effector domain; TNCB, 2.4.6-trinitro-1-chlorobenzene; TNBS, 2,4,6-trinitrobenzenesulfonic acid; sFasL, soluble FasL; TNP, 2,4,6-trinitrophenyl; TRAF, TNFR-associated factor.
Received for publication April 20, 2001. Accepted for publication July 10, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T lymphocytes for apoptosis. Nature 353:85.
B: TNF is not needed for induction of a biological effect via TNF receptors. J. Biol. Chem. 265:22409.. J. Immunol. 140:53.
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 T. J. Kemp, A. T. Ludwig, J. K. Earel, J. M. Moore, R. L. VanOosten, B. Moses, K. Leidal, W. M. Nauseef, and T. S. Griffith Neutrophil stimulation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) results in the release of functional soluble TRAIL/Apo-2L Blood, November 15, 2005; 106(10): 3474 - 3482. [Abstract] [Full Text] [PDF]
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 B. Depuydt, G. Van Loo, P. Vandenabeele, and W. Declercq Induction of apoptosis by TNF receptor 2 in a T-cell hybridoma is FADD dependent and blocked by caspase-8 inhibitors J. Cell Sci., February 1, 2005; 118(3): 497 - 504. [Abstract] [Full Text] [PDF]
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 H.-Y. Qin, P. Chaturvedi, and B. Singh In vivo apoptosis of diabetogenic T cells in NOD mice by IFN-{gamma}/TNF-{alpha} Int. Immunol., December 1, 2004; 16(12): 1723 - 1732. [Abstract] [Full Text] [PDF]
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 J. A. Rutigliano and B. S. Graham Prolonged Production of TNF-{alpha} Exacerbates Illness during Respiratory Syncytial Virus Infection J. Immunol., September 1, 2004; 173(5): 3408 - 3417. [Abstract] [Full Text] [PDF]
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D. A. Murray and I. N. Crispe TNF-{alpha} Controls Intrahepatic T Cell Apoptosis and Peripheral T Cell Numbers J. Immunol., August 15, 2004; 173(4): 2402 - 2409. [Abstract] [Full Text] [PDF]
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 J. Y. Niederkorn, E. Mayhew, J. Mellon, and S. Hegde Role of Tumor Necrosis Factor Receptor Expression in Anterior Chamber-Associated Immune Deviation (ACAID) and Corneal Allograft Survival Invest. Ophthalmol. Vis. Sci., August 1, 2004; 45(8): 2674 - 2681. [Abstract] [Full Text] [PDF]
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M. Umemura, T. Kawabe, K. Shudo, H. Kidoya, M. Fukui, M. Asano, Y. Iwakura, G. Matsuzaki, R. Imamura, and T. Suda Involvement of IL-17 in Fas ligand-induced inflammation Int. Immunol., August 1, 2004; 16(8): 1099 - 1108. [Abstract] [Full Text] [PDF]
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