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Cutting Edge |
-Induced Suppression of B Lymphocyte Formation1
,
,
* First Department of Internal Medicine, Faculty of Medicine,
Medicine and Biosystemic Science and
Anatomical Pathology, Graduate School of Medical Sciences,
Department of Medical Technology, School of Health Sciences, and
¶ Department of Molecular and Cellular Biology, Laboratory of Embryonic and Genetic Engineering, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan;
|| Department of Immunology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan;
# Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Osaka, Japan; and
** Core Research for Engineering, Science, and Technology, Japan Science and Technology Corporation, Saitama, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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inhibits B lymphocyte development, and the nuclear
protein Daxx has been reported to be essential for this biological
activity. We show in this study that IFN- inhibits the clonal
proliferation of B lymphocyte progenitors in response to IL-7 in
wild-type, but not in tyk2-deficient, mice. In addition,
the IFN--induced up-regulation and nuclear translocation of
Daxx are completely abrogated in the absence of tyk2. Therefore,
tyk2 is directly involved in IFN- signaling for the induction and
translocation of Daxx, which may result in B lymphocyte growth arrest
and/or apoptosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mediates potent antiviral and antiproliferative activities on target
cells through its interaction with IFN- receptors. Janus
kinase (Jak)3 1 and Tyk2,
the nonreceptor tyrosine kinases of the Jak family, are associated with
IFN- receptors and play a pivotal role in transducing IFN-
signals (1, 2, 3). Activated Jaks phosphorylate
tyrosine residues on IFN- receptors, thereby recruiting Stat1 and 2,
and other signaling molecules to the activated receptor complex. Stat1
and 2 are in turn phosphorylated by Jaks, and these
activated Stats subsequently associate to form either Stat1 homodimers
or the transcription factor IFN-stimulated gene factor-3, which
then translocates to the nucleus to regulate gene expression
(4, 5). Tyk2 was identified as a novel protein kinase,
based on its ability to rescue signaling in mutated fibroblasts,
nonresponsive to IFN- (6). However, we and others have
shown using tyk2-deficient mice that tyk2 has a restricted
function and does not play a major role in IFN- signaling (7, 8). In contrast, Jak 1-null cells fail to respond to
IFN- (9). As for Stat function, in
Stat1-null mice all the examined IFN--induced responses,
including the induction of MHC class I molecules in T cells, NO
production by macrophages, and antimicrobial or antiviral activities
were rendered defective in the absence of Stat1 (10, 11).
Stat2-null mice also showed an increased susceptibility to
viral infection (12). Accordingly, the Jak1-Stat signaling
pathway is thought to be essential for IFN- signaling.
IFN- is a potent inhibitor of IL-7-dependent growth of early B cell
lineage progenitors, effectively aborting further B cell lineage
differentiation at the pro-B cell stage (13). Although
many IFN--induced responses are abrogated in Stat1-deficient mice
(10, 11), the inhibition of IL-7-dependent B lymphopoiesis
by IFN- is unaffected in the absence of Stat1 (14).
Using previously generated tyk2-deficient mice, we show in
this study that tyk2 is essential for IFN--induced B lymphocyte
growth inhibition, through the up-regulation and nuclear
translocation of Daxx, which was recently identified as playing an
important role in the IFN--mediated inhibition of B lymphopoiesis
(15).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation of tyk2-deficient mice has been previously described (7). Mice were housed and bred in the Kyushu University Animal Center (Fukuoka, Japan).
Abs and cytokines
Anti-Daxx Ab (M-112) was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Murine natural IFN- was obtained from Hayashibara
Biochemical Institute (Okayama, Japan).
CFU-IL7 colony assay
Bone marrow cells were prepared and suspended in a 1-ml assay
medium as previously described (16, 17). The semisolid
agar assay for CFU-IL-7 was done with 1 ng/ml IL-7 with or without 1000
U/ml IFN-. Numbers of colonies were counted after 6 days of
culture.
In vitro bone marrow cultures
Bone marrow cells from wild-type or tyk2-deficient
mice were prepared and stimulated by 1000 U/ml IFN- for the
indicated times. Cells were harvested and analyzed for the expression
of Daxx.
Generation of B cell precursors on ST-2 cells
We generated B cell precursors from bone marrow cells by
coculturing with ST-2 cells (18). ST-2 is a bone
marrow-derived stromal cell line that can support the generation of B
lymphocyte progenitor cells. ST-2 cells produce IL-7, and the
production of IL-7 from ST-2 is enhanced by close association with
hemopoietic cells. ST-2 cells were grown to 
80% confluence in a
10-cm dish. Bone marrow cells (1 x
106) from wild-type or tyk2-deficient
mice were cultured on ST-2 cells with 10 ml of RPMI containing 10% FCS
and 5 x 10-5 mol/L 2-ME. Ten days after
coculture, 1000 U/ml IFN- was added to the dish. Cells were
harvested at the indicated times after adding IFN-.
RT-PCR analysis
RNA was isolated from freshly harvested cells using Isogen
(Nippon Gene, Tokyo, Japan) and dissolved in diethyl
pyrocarbonate-treated water. First-strand cDNA synthesis from
RNA was conducted using poly(dT) and reverse transcriptase, following
protocols supplied by the manufacturer (Takara Biomedicals, Tokyo,
Japan). Target RNA (1 µg) was reverse-transcribed using 0.25 U AMV
Reverse Transcriptase XL (Takara Biomedicals) at 42°C for 30 min in
the presence of 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 5 mM
MgCl2, 1 mM dNTPs, 0.25 U RNase inhibitor, and
0.125 µM oligo(dT)-adaptor primer. Each 20-µl PCR contained 1 µl
of first-strand cDNA, 1 µM each primer (sense and antisense), 0.2 mM
dNTPs, 1.5 mM MgCl2, 1x polymerase buffer, and 2
U Taq polymerase (Takara Biomedicals). For evaluation of
Daxx expression, forward, 5'-CCCATGGCCACCGATGACAGCAT-3', and
reverse, 5'-AGGGTTAGGGCCCGACGCCTCACT-3', primers were used.
Expression of 
-actin was examined by using forward,
5'-TAGACTTCGAGCAGGAGGAGATG-3', and reverse,
5'-CGTACTCCTGCTTGCTGATCCA-3', primers. The PCR mixture was denatured in
a thermal cycler at 94°C for 3 min and then 25 cycles were performed,
each consisting of denaturing at 94°C for 1 min, annealing at 60°C
for 2 min, and extension at 72°C for 3 min (15).
Real-time PCR assay
Relative quantification of Daxx in cells was performed using real-time quantitative PCR, by a TaqMan assay on an ABI 7000 system. Real-time quantitative PCR used a cDNA template with the appropriate primers as follows: primers used to amplify murine Daxx were 5'-CATGAACTGGTGACCAGCTCTCT-3' and 5'-TGGCCACACTGGTCTTATAAATACA-3', with a murine Daxx probe, FAM-5'-TCCATCCCTGCTTCTCCAGACACCC-3'-TAMRA. Primers used to amplify murine GAPDH were 5'-ACGGCAAATTCAACGGCA-3' and 5'-AGATGGTGATGGGCTTCCC-3', with a murine GAPDH probe, FAM-5'-AGGCCGAGAATGGGAAGCTTGTCATC-3'-TAMRA. PCR amplifications were performed in a 50-µl volume, containing 1 µl of cDNA template, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 10 mM EDTA, 200 µM dNTPs, 3 mM MgCl2, 200 nM of each primer, 0.625 U AmpliTaq Gold, and 0.25 U AmpErase uracil N-glycosylase. Each amplification also contained 100 nM of the appropriate detection probe. Each PCR amplification was performed in duplicate, using the following conditions: 50°C for 2 min preceding 95°C for 10 min, followed by 40 cycles of amplification (95°C for 15 s, 60°C for 1 min). In each reaction, GAPDH was amplified as a housekeeping gene to generate a standard curve and correct for variations in target sample quantity. Relative copy number was calculated for each sample based on the standard curve after normalization to GAPDH content.
Western blotting
Cells from wild-type or tyk2-deficient mice were lysed in lysis buffer as previously described (19). Cell lysates were centrifuged at 12,000 x g for 15 min to remove debris. Total cell lysates were resolved by SDS-10% PAGE and transferred to a nitrocellulose membrane. Membranes were probed using anti-Daxx and anti-Stat1 Abs and visualized with the ECL detection system (Amersham, Uppsala, Sweden).
Immunocytochemistry
Cells centrifuged onto glass slides were fixed in cold methanol for 20 min and washed extensively in PBS before blocking with 10% normal goat serum and incubating overnight at 4°C either with rabbit polyclonal anti-Daxx Abs (Santa Cruz Biotechnology) or with nonimmune rabbit serum as a negative control. The slides were then incubated for 1 h with an Alexa Fluor 488-conjugated goat anti-rabbit IgG Ab. After washing in PBS, slides were mounted with coverslips in glycerol containing antifade reagent and propidium iodide, and were examined by confocal microscopy (LSM-GB2000; Olympus, Tokyo, Japan) (15).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mediated inhibition of
IL-7-dependent B lymphopoiesis
IL-7 induces the proliferation of B cell precursors. We used a
colony assay to evaluate the role of tyk2 on IL-7-dependent growth
of B lymphocytes. The absence of tyk2 did not have any effect on
the number of IL-7-induced colonies of B lymphocytes from bone marrow
cells (Fig. 1
). IFN- suppressed the
CFU-IL-7 from wild-type mice, but IFN--induced B lymphocyte growth
suppression was not observed in tyk2-deficient bone marrow
cells (Fig. 1).
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We next examined the expression and localization of Daxx following
IFN- treatment, as Daxx has recently been reported to be important
in IFN--mediated inhibition of B lymphopoiesis
(15).
First, we examined Daxx expression in bone marrow cells before and
after IFN- stimulation. RT-PCR assessment of Daxx transcript levels
showed that Daxx expression was increased in bone marrow cells from
wild-type mice by treatment with IFN- for 8 h (Fig. 2A). In contrast, Daxx
transcript levels only increased slightly after IFN- treatment of
bone marrow cells from tyk2-/- mice (Fig. 2A). Analysis of expression by real-time quantitative TaqMan
PCR revealed that Daxx mRNA levels in bone marrow cells from wild-type
mice were significantly increased by IFN- stimulation (Fig. 2B). In contrast to wild-type mice, IFN- increased Daxx
transcript levels more modestly in bone marrow cells from
tyk2-deficient mice (Fig. 2B).
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treatment. The expression of Daxx
protein in bone marrow cells from wild-type mice was enhanced by
treatment with IFN- for 24 h. In contrast to wild-type mice,
IFN- did not increase Daxx protein levels in bone marrow cells from
tyk2-deficient mice (Fig. 2
C).
As IFN--mediated inhibition of B lymphocyte progenitor clonal
proliferation in response to IL-7 was not observed in
tyk2-deficient mice, we enriched B cell precursors by
coculturing bone marrow cells with the IL-7-producing stromal cell line
ST-2. Ten days after coculture, >95% of cells were positive for B220
(data not shown). By using enriched B cell precursors from wild-type
mice or tyk2-deficient mice, we examined the time course of
induction of Daxx expression by IFN- (Fig. 3A). The induction of Daxx
mRNA was rapid, and began to decrease after 17 h of IFN-
stimulation. There was no difference in the time course of Daxx
induction by IFN- in B cell precursors from wild-type and
tyk2-deficient mice. The increase in Daxx transcript levels
by IFN- in B cell precursors from tyk2-deficient mice,
however, was much less than that from wild-type mice.
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augmented the expression of Daxx protein in B cell precursors
from wild-type mice, with maximal induction of Daxx protein observed
after 48 h of IFN- stimulation (Fig. 3B). Although
IFN- also augmented the expression of Daxx protein in B cell
precursors from tyk2-deficient mice, the level of induction
was much less than from wild-type mice.
Tyk2 is required for IFN--mediated nuclear localization of
Daxx
The IFN--triggered pathway of apoptosis involves
translocation of Daxx to nuclear bodies and the related suppression of
cell-cycle-related genes. To evaluate changes in the intracellular
localization of Daxx protein, B cell precursors obtained from
coculturing bone marrow cells with ST-2 cells were examined before and
after IFN- treatment by immunofluorescence confocal microscopy with
polyclonal anti-Daxx Abs (Fig. 4).
Daxx nuclear body translocation was induced by IFN- in wild-type B
cell precursors (Fig. 4B) as previously reported. In
contrast, Daxx protein was not concentrated within nuclear bodies in
tyk2-deficient B cell precursors following IFN- treatment
(Fig. 4D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, Jak1 and tyk2 are
initially phosphorylated, followed by Stat1 and Stat2
activation. The activated Stats associate to form either Stat1
homodimers or IFN-stimulated gene factor-3, and then bind to the
IFN-stimulated response element of IFN--inducible genes. Although
tyk2 was initially cloned as a molecule essential for transducing
IFN- signals (6), we and others have shown that the
absence of tyk2 either did not affect the activation of Stat1 by
IFN-, or only affected some IFN--induced biological effects, such
as NO production from macrophages (7, 8). Moreover, most
IFN--induced biological functions such as anti-viral capacity
and MHC class I expression were not affected in the absence of
tyk2 (7, 8). In contrast, all IFN--induced
responses examined were rendered defective in the absence of Stat1
(10, 11). Accordingly, the Jak1-Stat1 signaling pathway is
thought to be essential for IFN- signaling.
IL-7, an essential growth factor for B and T cell lymphopoiesis in
mice, stimulates the formation of B lymphocyte colonies from bone
marrow cells in vitro (20). There was no difference in the
number of CFU-IL-7 in bone marrow cells from wild-type and
tyk2-deficient mice, indicating that the number of B
lymphocyte progenitor cells was not affected in
tyk2-deficient mice. IFN- inhibited the IL-7 promoted
growth of early B cell lineage cells (13). The number of
CFU-IL-7 from wild-type bone marrow cells was decreased by the addition
of IFN-. The inhibition of IL-7-dependent B lymphopoiesis by IFN-
was almost abrogated in the absence of tyk2 (Fig. 1), although it
was reported to be unaffected in Stat1-deficient mice
(14). Accordingly, other signaling molecules, besides
Stat1, which is phosphorylated by IFN- regardless of the
absence of tyk2 (7), are thought to transduce the
IFN- signal inhibiting B lymphocyte growth.
Daxx was reported to be involved in the Fas and TGF-
apoptosis-signaling pathway (21, 22). Daxx was
originally cloned as a FasR-associated protein and binds specifically
to the death domain of the FasR, although Daxx itself lacks a death
domain (21, 23, 24). Fas belongs to the TNFR superfamily
and induces apoptosis upon receptor oligomerization
(25). There are two independent signaling pathways
downstream of the FasR (24), involving the adapter protein
Fas-associated death domain (25) and Daxx
(23). The activation of Fas-associated death domain
induces a protease cascade (26, 27), while that of Daxx
induces c-Jun N-terminal kinase activation, leading to
apoptosis (23). Overexpression of Daxx enhances
Fas-induced apoptosis (23), and the targeted
disruption of the Daxx gene in mice results in embryonic
lethality (28). Daxx is also involved in coupling TGF-R
signaling with components of the apoptotic machinery (22).
TGF- induces apoptosis in primary and cultured lymphocytes
(29). Daxx associates with the cytoplasmic domain of the
type II TGF-R and transduces apoptotic signals by TGF-
(22). The C-terminal portion of Daxx (resides 626–739)
acts as a dominant negative inhibitor of Fas-mediated
apoptosis, presumably by competing with endogenous Daxx for
binding to Fas. The expression of the C-terminal portion of Daxx
suppresses TGF--mediated apoptosis, as well as Fas-mediated
apoptosis (22).
Recently, Daxx was also reported to be essential for inhibition of B
lymphopoiesis by IFN- (15). IFN- enhances Daxx
expression, with concomitant increases in Daxx protein levels and
nuclear body translocation. Moreover, Daxx antisense oligonucleotides
rescue IFN--treated pro-B cells from growth arrest and
apoptosis. Therefore, we examined the expression of Daxx mRNA
and protein in tyk2-deficient cells. Strikingly, the
augmentation of Daxx expression by IFN-, which is observed in
wild-type cells, was drastically reduced in tyk2-deficient
cells (Figs. 2 and 3). Moreover, the nuclear translocation of Daxx by
IFN- was also abrogated in the absence of tyk2 (Fig. 4). Our
study demonstrates that tyk2 is essential for the transduction of
IFN--induced B lymphocyte growth arrest signals through the
activation of some signaling molecule other than Stat1, followed by the
up-regulation and nuclear translocation of Daxx. To clarify the
biochemical interactions between tyk2 and Daxx, we examined
whether tyk2, which is a tyrosine kinase and activates Stats
through phosphorylation, also
phosphorylated Daxx. Daxx was not
phosphorylated in B lymphocytes stimulated with IFN-
(data not shown). In addition, no changes in the
phosphorylation pattern of Daxx were detected upon
stimulation with TGF- or Fas (22).
The apoptotic signaling pathway downstream of Daxx is as yet unknown.
Because Daxx associates with Fas and TGF-RII, it may also associate
with the IFN-R, tyk2, or with the IFN-R/tyk2 complex.
The presence of Daxx within the receptor complex may be important for
the recruitment and activation of the next substrate downstream of Daxx
in the Fas, TGF-, and IFN- apoptosis-signaling pathway.
Alternatively, the localization of Daxx may be important. Although Daxx
is an adapter protein and is associated with Fas and TGF-RII, it can
localize to the nucleus of some cells (30). The
localization of Daxx in either the cytoplasmic or nuclear compartment
was reported to be dependent upon the cell type and/or its functional
status (31). The subcellular distribution of the Daxx
protein was not changed even after exposure of the cells to TGF-
(22). In contrast, the IFN--triggered pathway of
apoptosis involved the translocation of Daxx to nuclear bodies
and the related suppression of cell cycle-related genes
(15). The requirement of tyk2 for IFN--mediated
apoptosis of B lymphocytes may be a direct result of the
inhibition of Daxx nuclear translocation by IFN- (Fig. 4).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kazuya Shimoda, First Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan. E-mail address: kshimoda{at}intmed1.med.kyushu-u.ac.jp
3 Abbreviation used in this paper: Jak, Janus kinase.
Received for publication May 20, 2002. Accepted for publication September 4, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ and -
signal transduction. Nature 366:129.[Medline]
signal transduction pathway. Nature 366:166.[Medline]
/ signaling pathway. Cell 70:313.[Medline]
signaling, although it is required for IL-12-mediated T cell function. Immunity 13:561.[Medline]
-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation. Nat. Cell Biol. 3:708.[Medline]
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