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by Activated Th1 Cells Occurs Via Reverse Signaling Through TNF-Related Activation-Induced Cytokine1
*
Department of Microbiology and Immunology, and
Immunology Research Center, National Yang-Ming University, Taipei, Taiwan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B (RANK).Fc fusion
protein suppresses IFN-
secretion by activated Th1 cells, but does
not affect IL-4 secretion by Th2 cells. The suppressive effect on
IFN- secretion is observed when Th1 cells are activated by APCs, but
not by immobilized anti-TCR
mAb. In contrast, immobilized
RANK.Fc fusion protein augments IFN- secretion by Th1 cells,
indicating the occurrence of reverse signaling through TRANCE during T
cell/APC interaction. The enhanced secretion of IFN- mediated via
TRANCE correlates with the activation of p38 mitogen-activated protein
kinase and is blocked by SB203580, a p38 mitogen-activated protein
kinase-specific inhibitor. Thus, in addition to its role in activating
dendritic cells by binding to the receptor RANK, TRANCE itself can
signal the augmentation of IFN- secretion via a p38-dependent
pathway, and this provides yet another example of reverse signaling by
a member of TNF superfamily. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (RANK) ligand
(1), is a member of TNF superfamily that is induced upon
TCR engagement and activates c-Jun N-terminal kinase after interaction
with its receptor RANK (1, 2, 3). TRANCE augments the ability
of dendritic cells (DCs) to stimulate naive T cell proliferation in a
MLR and increases the survival of RANK+ T cells
(2). In addition, a recent study demonstrated that TRANCE
is able to enhance the longevity and adjuvant properties of DCs in vivo
(4). Recently, a cytokine secreted by osteoblasts/stromal
cells, osteoprotegrin ligand (OPGL) (5, 6) (also named as
osteoclast differentiation factor (7)), was found to be
identical with TRANCE, and short-term administration of soluble rTRANCE
into normal adult mice results in osteoclast activation associated with
systemic hypercalcemia. Therefore, TRANCE also plays a role in the
activation of isolated mature osteoclasts in vitro. However, this
effect is inhibited by a soluble TNFR-like molecule,
osteoclastogenesis-inhibitory factor (also known as osteoprotegrin
(OPG) or osteoclast differentiation factor receptor) (5, 6). Thus, TRANCE seems to be an important regulator not only in
osteoclastogenesis, but also in the immune system, via its interaction
with RANK and OPG.
TRANCE is primarily expressed in T cells, T cell lines, and lymphoid
tissues. Upon TCR/CD3 stimulation, TRANCE mRNA and surface protein
expression are rapidly up-regulated in CD4+ and
CD8+ T cells, and this can be further enhanced in
CD4+ T cells by CD28-mediated costimulation
(8). In contrast to TRANCE, RANK is ubiquitously expressed
in human tissues. High levels of RANK expression are found in mature
DCs, while T and B cells only express low levels of RANK after
activation. In DCs, TRANCE induces the expression of proinflammatory
cytokines (IL-1, IL-6) and T cell growth and differentiation factors
(IL-12, IL-15), in addition to enhancing DC survival
(1, 2, 3). Moreover, TRANCE cooperates with CD40 ligand
(CD40L) or TNF-
to further increase the viability of DCs
(4), suggesting that these TNF-related molecules are
important in the activation of DCs and in modulating the immune
system.
Recently, our understanding of the signaling pathways downstream of members of TNFR superfamily has advanced dramatically. In addition, there is growing evidence that ligands of the TNF superfamily, such as CD40 ligand (CD40L/CD154) (9, 10, 11), CD30L (12), CD27 ligand (CD27L/CD70) (13), FasL (14, 15), CD137L (16), and OX40L (17), also transduce signals after engagement with their receptors. It has been shown that reverse signaling via CD40L is involved in a range of different immune processes, such as cytokine production, costimulation of T cell activation, and proper formation of germinal centers (10). Blair et al. (11) also demonstrated that CD40L could trigger short-term CD4+ T cell activation as well as mediating the secretion of immunomodulatory cytokines and apoptosis. In addition, Wiley et al. (12) showed that cross-linking of CD30L by a mAb or by CD30.Fc fusion protein induced the production of IL-8 by freshly isolated neutrophils. Moreover, reverse signaling via CD27L/CD70 has been shown to induce a subset of leukemic B cells to proliferate vigorously, an effect that is synergistically enhanced by ligation of CD40, but inhibited by the presence of IL-4 (13). Recently, Suzuki and Fink (14, 15) further demonstrated that maximal proliferation of CTL requires reverse signaling through FasL. It is also interesting to note that addition of CD137.Fc fusion protein induces a substantial degree of proliferation in human peripheral monocytes (16). Furthermore, cross-linking of OX40L on CD40L-stimulated B cells results in a significantly enhanced proliferative response of B cells and the down-regulation of the transcription factor B cell lineage-specific activator protein (17). These studies provide evidence to demonstrate the importance of reverse signaling in the activation of the immune system. Therefore, it is interesting to know whether bidirectional signaling might also occur in other members of TNFR superfamily.
In this study, we report that blockade of the interaction between T
cells and APCs by soluble RANK.Fc fusion protein suppresses the
secretion of IFN- by activated T cells. However, RANK.Fc fusion
protein does not exert any effect when T cells are activated by
immobilized anti-TCR mAb. In addition, cross-linking of TRANCE
by RANK.Fc enhances the secretion of IFN- in activated, but not
resting, CD4+ cells in a dose-dependent manner.
IL-12 is not required for the enhanced secretion of IFN- by
activated T cells, but p38 mitogen-activated protein kinase (MAPK) is
apparently activated after the engagement of TRANCE. Thus, it appears
that reverse signaling is also occurring following the interaction of
TRANCE and RANK. This provides yet another example of reverse signaling
by a member of TNF superfamily.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Receptor-Fc fusion protein was produced as previously described (18). The coding sequence for the extracellular domain of human RANK was isolated by RT-PCR using the forward primer, 5'-CGGGATCCACCACCATGGCCCCGCGCGCC-3', and the reverse primer, 5'-CGGGATCCATGGGGTTCATTTGGTGGTTT-3'. The amplified product was ligated in-frame into BamHI-cut pUC19-IgG1-Fc vector containing the human IgG1 Fc coding sequence. The fusion gene was then subcloned into pBacPAK9 vector (Clontech, Palo Alto, CA). RANK.Fc protein was recovered from the filtered supernatants of the recombinant virus-infected Sf21 cells using protein A-Sepharose beads. The bound RANK.Fc protein was eluted with glycine buffer (pH 3) and dialyzed into PBS.
Mice
The TCR transgenic mice AND (on a B10A
(H-2a) background) and IL-12 p35 gene knockout
mice (on a 129 background) were kindly provided by Dr. John Kung (IMB,
Academia Sinica, Taipei, Taiwan). The AND mice express a TCR
(V11/V3) transgene recognizing residues 88–103 of moth
cytochrome c (MCC) in the context of
I-Ek. The AND transgene was screened for by
anti-V3 mAb (clone KJ-25) staining and flow cytometry analysis.
All mice were maintained in the animal center at the National Yang-Ming
University and were used between 8 and 12 wk of age. All experiments
were performed in accordance with our institutional guidelines.
Separation of CD4+ T cells
CD4+ lymph node cells were purified by magnetic separation using MACS (Miltenyi Biotec, Bergisch Gladbach, Germany). The lymph node cells were incubated with FITC-labeled anti-CD4 mAbs for 25 min at 4°C, followed by washing with cold PBS and subsequent incubation with anti-FITC-conjugated microbeads. Briefly, 107 cells in 90 µl buffer (PBS with 2 mM EDTA) were incubated with 10 µl anti-FITC-conjugated microbeads for 15 min, and the CD4+ cells were isolated using a magnet (VarioMACS; Miltenyi Biotec), according to the vendor’s instructions. The purity of sorted CD4+ cells was determined by flow cytometry.
In vitro activation of T lymphocytes
CD4+ lymph node cells (5 x
105/0.5 ml/well of a 24-well plate) from AND TCR
transgenic mice were activated by MCC88–103
peptide (10 µg/ml)-pulsed, mitomycin C (50 µg/ml)-treated
splenocytes (I-Ek) in vitro for 5 days.
Alternatively, CD4+ T cells were activated by
plate-bound-anti-TCR mAb (clone H57.597, 1.25 µg/well) in
vitro for 5 days. After initial stimulation, CD4+
T cells were transferred to fresh plates for 24 h before
subsequent stimulation by MCC88–103 peptide (10
µg/ml)-pulsed, mitomycin C (50 µg/ml)-treated splenocytes
(I-Ek), or plate-bound Ab. All cells were
cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY)
supplemented with penicillin (100 U/ml; Sigma, St. Louis, MO),
streptomycin (100 mg/ml; Sigma), 50 µM 2-ME, and 10% FCS for 5 days.
IL-2 (500 U/ml; R&D Systems, Minneapolis, MN) was added to the culture
supernatant every 3 days to maintain cell growth. Anti-IL-4 mAb (clone
BVD4, 10 µg/ml) was added to the culture supernatant at the priming
stage to induce T cell differentiation to a Th1 phenotype, while IL-4
(400 U/ml; R&D Systems), anti-IFN- mAb (clone R4-6A2, 10
µg/ml), and anti-IL-12 antiserum (R&D Systems) were added at the
priming stage to induce T cell differentiation to a Th2 phenotype. A
total of 100 µl/well of supernatant was removed and replaced with
same amount of fresh medium containing all supplements at 72 h
after initial stimulation to maintain cell growth.
To trigger the activation of T cells via TRANCE,
CD4+ T cells from AND TCR transgenic mice were
stimulated with Ag-pulsed APCs, in the presence of IL-2 and
anti-IL-4 mAb for 5 days. Alternatively, CD4+
T cells from either AND or BALB/c mice were stimulated with plate-bound
anti-TCR mAb, in the presence of IL-2 and anti-IL-4 mAb for
5 days. After resting on noncoating plate with fresh medium for 24
h, the CD4+ T cells were restimulated with
plate-bound anti-TCR mAb (1.25 µg/0.5 ml/well), in conjunction
with immobilized RANK.Fc fusion protein (20 µg/well) or human IgG1
(20 µg/well; Sigma). Supernatants were separated from cells by
centrifugation, and cytokine content was determined by ELISA, while the
cell pellets were used for kinase assays.
For the determination of kinase activity, cells were washed with cold PBS twice, then lysed with cold buffer A (20 mM Tris-HCl, pH 8, 200 mM NaCl, 10% glycerol, 1 mM PMSF, 2 µg/ml leupeptin, 2 µM/ml aprotonin, 0.1% 2-ME, 0.5% Nonidet P-40). The suspension lysates were separated and stored at -70°C for use in kinase assays.
Flow cytometry analysis and Abs
The detection of intracellular cytokines was performed as
described previously (19). Before staining, cells were
incubated with anti-mouse CD32/CD16 mAbs (1
µg/106 cells, clone 2.4G2) at 4°C for 10 min
to prevent nonspecific binding. Samples were then stained with
biotin-labeled RANK.Fc fusion protein (1 µg/106
cells), followed by CyChrome-conjugated streptavidin (PharMingen, San
Diego, CA) to detect the expression of TRANCE. To detect the
intracellular IFN-, the same samples were fixed with 1%
paraformaldehyde in PBS at 4°C for 15 min, followed by incubation
with FITC-conjugated anti-mouse IFN- (clone XMG1.2) in 1%
FCS/0.1% (w/v) NaN3 (Sigma)/0.1% (w/v) Saponin
(Sigma)/PBS at 4°C for 30 min. Finally, cells were washed and
analyzed by FACSvantage (Becton Dickinson, San Jose, CA) using
CellQuest software (Becton Dickinson). All the Abs used for flow
cytometry analysis were purchased from PharMingen.
Enzyme-linked immunosorbent assay
Cell culture supernatants were collected at various intervals,
and levels of murine IFN-, IL-4, IL-12, IL-18, and TNF- were
quantitated using commercial ELISA kits (R&D Systems), according to the
vendor’s instructions.
p38 Kinase activity assay
Specific protein kinase was immunoprecipitated from cell lysates
by incubation for 2 h at 4°C with anti-p38 polyclonal Ab
(Bio-Lab, St. Paul, MN) and protein A/G beads (Pharmacia, Piscataway,
NJ). The immunoprecipitates were washed twice with buffer A and then
twice with kinase buffer (25 mM HEPES, pH 7.4, 25 mM
-glycerophosphate, 25 mM MgCl2, 0.5 mM DTT,
0.1 mM sodium orthovanadate). Protein kinase assays were performed by
the incubation of the immunoprecipitates with 1 µg of substrate
(myelin basic protein, MBP) and 50 µM
[-32P]ATP (10 Ci/mmol) in a final volume of
40 µl of kinase buffer for 30 min at 30°C. The reactions were
terminated by addition of Laemmli sample buffer, and the
phosphorylation status of MBP was visualized by autoradiography after
fractionation on SDS-PAGE.
Western blot analysis
Cell lysates (7.5 x 105 cells/sample) were fractionated by SDS-PAGE, then were transferred to nitrocellulose paper (Hybond-C extra; Amersham, Arlington Heights, IL) by electrophoresis at 0.8 mA/cm2 for 1.5 h at room temperature. Blots were incubated with blocking buffer (5% fat-free skimmed milk/PBS) at 4°C for 12–16 h before probing. Specific anti-p38 polyclonal Ab (Bio-Lab) is diluted in the blocking buffer, and the incubation was conducted at room temperature with constant agitation for1 h. The blot was washed and incubated with HRP-conjugated secondary Ab diluted in blocking solution for 45 min. After wash with PBS, membranes were dried and developed with an ECL system (Amersham) for 1 min, then exposed to x-ray film.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the expression of TRANCE on the differentiated T cells
(Th1 or Th2), we constructed a soluble fusion protein comprising the
extracellular domain of human RANK and the Fc domain of human IgG1
(Fig. 1
A). To determine the
expression of TRANCE on activated T cells, CD4+
lymph node cells isolated from TCR transgenic AND mice were cultured in
vitro under conditions favoring differentiation into Th1 or Th2
subsets. The AND transgenic mice express the V11V3 transgene from
a TCR specific for MCC88–103 in the context of
I-Ek. After priming, T cells were restimulated by
anti-TCR mAb, and the expression levels of surface TRANCE and
secreted or intracellular cytokines (IFN- and IL-4) were determined.
A significant shift in TRANCE-associated fluorescence was observed at
24 h after restimulation in Th1 cells, but not in Th2 cells (Fig. 1B, top). The surface expression of TRANCE was in
accordance with the expression of TRANCE mRNA in Th1 and Th2 subsets
(Fig. 1B, lower left). We then asked whether
TRANCE and IFN- were coexpressed by the same Th1 cells. At 12 h
after restimulation, 31.25% expressed both TRANCE and intracellular
IFN-. These TRANCE+
IFN-+ double-positive cells reached peak at
18 h. Therefore, a large proportion of
TRANCE+ cells is also actively producing IFN-
at 18 h. After 24 h, most of intracellular IFN- was
secreted into the supernatant under the stimulation conditions used.
Therefore, it was difficult to study the coexpression of TRANCE and
IFN- at this time point.
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secretion by Th1
cells
We then investigated the role of TRANCE in IFN- secretion
during T cell activation. To address this question, AND
CD4+ T cells were stimulated with
MCC88–103-pulsed APCs, either in the presence or
absence of soluble rRANK.Fc fusion protein. After resting, cells were
restimulated by MCC88–103-pulsed APCs for
24 h, and the cytokines secreted by T cells were quantitated by
ELISA. We found that secretion of IFN- was dramatically suppressed
by soluble RANK.Fc fusion protein, when T cells were primed in
conditions favoring Th1 differentiation (Fig. 2A, top left). In
contrast, RANK.Fc fusion protein only had a minimal effect on IL-4
secretion by Th1 or Th2 cells (Fig. 2A, top
right). This observation suggests that inhibition of TRANCE/RANK
interactions during priming suppresses the ability of Th1 cells
to secrete IFN- following subsequent stimulation. Because IL-12 and
IL-18 have been shown to be potent inducers for IFN- secretion
(20, 21), we asked whether blockade of TRANCE/RANK
interactions during priming affected the secretion of IL-12 and IL-18
by APCs. However, the levels of IL-12 and IL-18 in the culture
supernatant were not significantly affected when cells were cultured in
the presence or absence of RANK.Fc fusion protein during priming (Fig. 2A, bottom). Thus, the suppression of IFN-
secretion is not due to the depressed IL-12 and IL-18 secretion
associated with mitomycin-treated APCs.
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mAb in place of Ag-pulsed
APCs to elicit T cell stimulation, both in the priming and
restimulation stage. Surprisingly, the secretion of IFN- by
CD4+ T cells was not significantly affected by
RANK.Fc when T cells were activated by immobilized anti-TCR mAb
(Fig. 2
B). Therefore, the inhibitory effect of RANK.Fc
during T cell activation is dependent on the presence of APCs,
suggesting the importance of TRANCE/RANK interactions between T cells
and APC during T cell activation. Reverse signaling via TRANCE
From the above experiments, it is clear that the interference of
TRANCE/RANK interactions can suppress IFN- secretion. However, the
effect is not due to the altered production of IL-12 and IL-18 by
macrophages. Because members of TNF superfamily, e.g., CD40L
(9, 10, 11), CD30L (12), CD27L (13),
FasL (14, 15), CD137L (16), and OX40L
(17), have been shown to deliver reverse signals, and thus
modulate the activation of T cells or neutrophils, we asked whether the
phenomenon observed in this study results from the blockade of
signaling transduced by TRANCE on T cells, rather than by RANK on
macrophages. To answer this question, Th1 cells from AND TCR transgenic
mice, which had been incubated in the presence or absence of
immobilized RANK.Fc fusion protein at the priming stage, were
restimulated with immobilized anti-TCR mAb, and IFN-
secretion was measured. We found that cross-linking of TRANCE alone had
no effect on the production of IFN-. In contrast, when both TCR and
TRANCE were cross-linked by anti-TCR mAb (1.25 µg/well) and
RANK.Fc (20 µg/well), respectively, the secretion of IFN- was
enhanced dramatically (Fig. 3A). The increase in IFN-
production was proportional to the increased concentration of
plate-bound RANK.Fc in a dose-dependent manner (Fig. 3B).
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mAb with or without plate-bound RANK.Fc,
both in the priming and restimulation stages. Among the four groups
tested, we found that the highest level of IFN- production was
produced when Th1 cells were cross-linked by anti-TCR mAb and
RANK.Fc fusion proteins in both the priming and restimulation stages.
The levels of IFN- produced, from highest to lowest, were
RANK.Fc/RANK.Fc > RANK.Fc/IgG1 > IgG1/RANK.Fc >
IgG1/IgG1, for both BALB/c (Fig. 3C) and AND T cells (Fig. 3D). This observation suggested that engagement of TRANCE
could transduce a costimulatory signal to enhance IFN- secretion
during both priming and restimulation, and this phenomenon can be
observed in both TCR transgenic AND as well as non-TCR transgenic
BALB/c mice (Fig. 3, C and D). In contrast, the
different combinations of reagents at the priming and restimulation
stages had no significant effect on IL-4 production by Th2 cells (Fig. 3, C and D).
It has been shown that TRANCE induces the expression of proinflammatory
cytokines (IL-1, IL-6) and T cell growth and differentiation factors
(IL-12, IL-15) in DCs (8). To further confirm that the
enhanced secretion of IFN- via TRANCE is not dependent on IL-12 in
this assay system, CD4+ T cells isolated from
IL-12 knockout mice were stimulated with immobilized anti-TCR mAb
and RANK.Fc, after which the level of secreted IFN- in the culture
supernatant was measured. As shown in Fig. 3E, highest level
of IFN- production was observed when the cells were cross-linked by
anti-TCR mAb and RANK.Fc fusion proteins during both the priming
and restimulation stages. This observation provides unequivocal
evidence for the existence of an IL-12-independent pathway for the
enhanced IFN- secretion via a costimulatory signal triggered by the
cross-linking of TRANCE.
Engagement of TRANCE activates p38 MAPK
We wished to understand the signaling pathway transduced by
TRANCE. To address this question, T cells activated by plate-bound
anti-TCR mAb and RANK.Fc were incubated with SB203580 (p38 MAPK
inhibitor), PD98059 (mitogen-activated protein/extracellular
signal-related kinase kinase inhibitor), rapamycin (p70 S6 kinase
inhibitor), and D609 (phosphatidylcholine-specific phospholipase
inhibitor). Among these kinase inhibitors, only SB203580 could suppress
the secretion of IFN- in a dose-dependent manner (Fig. 4A). However, SB203580 did not
affect the secretion of TNF- from activated T cells. In contrast,
PD98059, rapamycin, and D609 had only minimal effects on IFN-
secretion (Fig. 4A). To demonstrate that the engagement of
TRANCE activates p38 MAPK directly, we precipitated p38 MAPK from
activated T cells to determine its activity (Fig. 4, B and
C). Compared with T cells cross-linked with anti-TCR
mAb alone, p38 MAPK activity was increased in T cells activated by
immobilized anti-TCR mAb and RANK.Fc in a dose-dependent manner
(Fig. 4C). The p38 MAPK activity increased from 1.5 h
and reached peak at 24 h after stimulation. Treatment of SB203580
suppressed the p38 MAPK activity induced by TRANCE-mediated reverse
signaling (lanes 5, 8, and 9 in
Fig. 4C). This provided direct evidence that the engagement
of TRANCE enhances the secretion of IFN- via the activation of p38
MAPK, with the p38 MAPK inhibitor SB203580 completely suppressing the
up-regulation of IFN- secretion via TRANCE.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-4, IL-5, and IL-6 is
significantly reduced in purified opgl-/- T
cells (6). Both Th1 and Th2 helper cell subtypes developed
in the absence of OPGL expression, but production of both Th1 and Th2
cytokines was significantly reduced in opgl-/-
T cells. These data indicate that OPGL has no apparent role in the
Th1/Th2 dichotomy, but is required for optimal cytokine production
following Ag-receptor activation. Our study has demonstrated that
RANK.Fc inhibits the secretion of IFN- by Th0 or Th1 cells, but not
by Th2 cells. This effect does not result from the direct inhibition of
IL-12 and IL-18 secretion by macrophages, because the levels of IL-12
and IL-18 are not changed significantly. However, we have demonstrated
that triggering of TRANCE by immobilized RANK.Fc, in conjunction with
immobilized anti-TCR mAb, activates p38 MAPK to enhance IFN-
secretion by activated CD4+ T cells. Thus, the
decrease of IFN- production in our assay system might result from
the inhibition of p38-dependent reverse signaling by TRANCE on
CD4+ T cells. This observation provides an
explanation for the reduced production of IFN- by
opgl-/- T cells, which lack cell surface TRANCE
and, therefore, cannot transduce the signal required to activate p38
MAPK and hence IFN- secretion. This could also explain why the
addition of soluble rTRANCE cannot restore the secretion of IFN- by
opgl-/- T cells to the same level as that of
wild-type T cells (6).
Even though IL-12 is not required for activation of p38 MAPK via
reverse signaling through TRANCE, we cannot rule out the possibility
that other IFN--modulatory factors secreted by macrophages are also
impaired by soluble RANK.Fc. Prehn et al. (22) have
reported that a soluble factor, which is distinct from IL-12 and IL-18,
produced by lamina propria mononuclear cells is required for
enhancement of IFN- production by CD4+ T
cells. Therefore, although the levels of IL-12 and IL-18 are not
affected by the addition of RANK.Fc in our assay system, it is possible
that other IFN--modulatory factors may be involved in the reduction
of IFN- production by CD4+ T cells.
Many cellular functions of p38 MAPK have been defined through the use
of specific p38 kinase inhibitors, the pyridinyl imidazoles. One such
compound, SB203580, blocks the activity of p38 and p38, but does
not inhibit p38 and p38
(23), nor the extracellular
signal-related kinases or c-Jun N-terminal kinases (24, 25). It has been reported that the production of IFN- by T
cells, following activation by specific Ag-pulsed APC, is mediated by
the p38 MAPK signaling pathway (26). Therefore, our
observation is in accord with previous observations and further
supports the essential role of p38 MAPK in IFN- production by
activated T cells.
Even though the phenomenon of reverse signaling has been observed in several members of TNF superfamily, including CD40L/CD154, CD30L, CD27L/CD70, FasL, CD137L, and OX40L (9, 10, 11, 12, 13, 14, 15, 16, 17), however, the downstream signaling pathways after the cross-linking of TNF and other members of TNF family have not been elucidated until recently. It has been reported that a casein kinase I (CKI) consensus sequence is conserved in the cytoplasmic domain of 6 of 15 members of the type II integral membrane TNF ligand family (27). Therefore, Watts et al. (27) speculated that the CKI motif might be also phosphorylated in other TNF ligand family member. This represents a new insight into the mechanism of reverse signaling in this cytokine family. However, there is no CKI motif in the cytoplasmic region of TRANCE, and our study provides the first evidence that p38 MAPK is involved in reverse signaling via TRANCE. This raises the question as to whether MAPK signaling pathways are also initiated via other members of TNF superfamily.
The presence of reverse signaling further increases the complexity to
our current understanding of TNF/TNFR superfamilies. If the reverse
signaling is a general phenomenon for all the members of TNF
superfamily, then it would be interesting to test whether cross-linking
of TNF-related apoptosis-inducing ligand (TRAIL) can also activate
signaling cascade. To date, four homologous human receptors for TRAIL
have been identified, including DR4 (28),
DR5/TRAIL-R2/TRAIL receptor inducer of cell killing 2 (29, 30), TRAIL receptor without an intracellular domain/decoy
receptor 1 (DcR1)/TRAIL-R3 (29, 31), and TRAIL-R4/DcR2
(32, 33), called TRAIL-R1, -R2, -R3, and -R4,
respectively. Among the four TRAIL receptors, the DcR1/TRAILR3 and
DcR2/TRAILR4 do not contain death domain and are unable to transduce
death signal like TRAILR1/DR4 and TRAILR2/DR5. However, DcR1 and DcR2
might be able to cross-link TRAIL to transduce the reverse signal.
Thus, DcR1 and DcR2 might play another role in the reciprocal signaling
between TRAIL and TRAIL receptors. Moreover, two soluble receptors of
the TNFR superfamily, OPG and DcR3, have been identified as dimeric
proteins, respectively (34, 35). The OPG has been shown to
bind to TRANCE (7) and TRAIL (36), while DcR3
has been demonstrated to interact with FasL (35) and LIGHT
(homologous to lymphotoxins, exhibits inducible expression, and
competes with HSV glycoprotein D for HVEN, a receptor expressed by T
lymphocyte) (37); thus, it would be interesting to test
whether OPG and DcR3 could bind to TRANCE/TRAIL and FasL/LIGHT to
trigger signaling cascade in cells expressing these ligands. In our
preliminary study, we found that both the plate-bound and soluble
OPG.Fc can suppress the secretion of IFN- from
anti-TCR-activated T cells (unpublished data). This raised the
possibility that the soluble receptors of TNFR superfamily might be
able to transduce signals by cross-linking their ligands expressed on
cell surface, and cannot be only regarded as decoy receptor to inhibit
the interaction between membrane form ligand and receptor. Recently,
Eissner et al. (38) demonstrated that reverse signaling
through transmembrane TNF conferred resistance to LPS in human
monocytes and macrophages by down-regulation of LPS-induced soluble TNF
and IL-6 as well as IL-1 and IL-10. Based on this information, the
shedding of TNFR during inflammation (39) might have the
potential to bind to the membrane form TNF to down-regulate
inflammation reaction.
In conclusion, our result provides another evidence to demonstrate the existence of reverse signaling in a member of TNF superfamily, suggesting bidirectional signaling might be a general phenomenon in ligand/receptor interactions of TNF/TNFR superfamilies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Shie-Liang Hsieh, Department and Institute of Microbiology and Immunology, National Yang-Ming University, Shie-Pai, Taipei 11221, Taiwan.
3 Abbreviations used in this paper: TRANCE, TNF-related activation-induced cytokine; CKI, casein kinase I; DC, dendritic cell; DcR, decoy receptor; L, ligand; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MCC, moth cytochrome c; OPG, osteoprotegrin; OPGL, osteoprotegrin ligand; RANK, receptor activator of NF-B; TRAIL, TNF-related apoptosis-inducing ligand.
Received for publication April 3, 2000. Accepted for publication October 10, 2000.
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References |
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