Download Critical regulation of CD4 T cell survival and autoimmunity by b

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Critical regulation of CD4+ T cell survival and
autoimmunity by b-arrestin 1
Yufeng Shi1, Yan Feng2, Jiuhong Kang1, Chang Liu1, Zhenxin Li3, Dangsheng Li4, Wei Cao2, Ju Qiu2,
Zhengliang Guo5, Enguang Bi1, Lei Zang1, Chuanzhen Lu3, Jingwu Z Zhang2,5,6 & Gang Pei1
CD4+ T cells are important in adaptive immunity, but their dysregulation can cause autoimmunity. Here we demonstrate that the
multifunctional adaptor protein b-arrestin 1 positively regulated naive and activated CD4+ T cell survival. We found enhanced
expression of the proto-oncogene Bcl2 through b-arrestin 1–dependent regulation of acetylation of histone H4 at the Bcl2
promoter. Mice deficient in the gene encoding b-arrestin 1 (Arrb1) were much more resistant to experimental autoimmune
encephalomyelitis, whereas overexpression of Arrb1 increased susceptibility to this disease. CD4+ T cells from patients with
multiple sclerosis had much higher Arrb1 expression, and ‘knockdown’ of Arrb1 by RNA-mediated interference in those cells
increased apoptosis induced by cytokine withdrawal. Our data demonstrate that b-arrestin 1 is critical for CD4+ T cell survival
and is a factor in susceptibility to autoimmunity.
Apoptosis of T cells is tightly controlled to maintain proper immune
homeostasis. Selected by means of T cell receptor signaling in the
thymus, most T cells die, and those remaining enter the peripheral
lymphoid organs and form the peripheral T cell repertoire. Peripheral
CD4+ T cells are maintained in a homeostatic balance between their
production and their elimination1,2. After antigen recognition, naive
CD4+ T cells proliferate and differentiate into effector T cells to
regulate immune responses. When the immune response is reduced,
several distinct pathways, including cell inactivation, activationinduced cell death and activated T cell–autonomous death, are used
to quell the number and activity of effector T cells. In all of these
processes, failure of CD4+ T cell death caused by incorrectly expressed
death-signaling regulators may either prevent the apoptosis of potentially autoreactive CD4+ T cells or prolong CD4+ T cell immune
responses, both of which can result in disturbed T cell homeostasis
and immune diseases such as autoimmunity and leukemogenesis3–6.
In mammals, CD4+ T cell death is mediated mainly by two distinct
signaling pathways1,6. One pathway is induced by cell surface receptors
such as TNF, TRAIL receptor and Fas, which are death factors for
activated CD4+ T cells; signaling through these receptors directly
activates apoptosis through the recruitment and activation of caspase
enzymes6. In addition to the death-receptor pathway, the Bcl-2 protein
family regulates T cell apoptosis through a separate mitochondriamediated pathway. The Bcl-2 protein family is composed of pro- and
antiapoptotic members that regulate apoptosis by controlling the
integrity of mitochondrial membranes and the release of proapoptotic
molecules that reside in the mitochondria7,8. Proteins of the Bcl-2
family are critical regulators of cytokine withdrawal–induced and
stress-induced T cell apoptosis, as well as autonomous death of
activated T cells1,9–12. Bcl-2 is the prototypic member of this large
protein family. In Bcl-2-deficient mice, T cells are much more
susceptible to death, and leukopenia occurs as these mice grow
old13. Conversely, in Bcl2-transgenic mice, T cells are more resistant
to many apoptotic stimuli, immune responses10,11 are prolonged, and
some autoimmune symptoms have been reported14.
The two b-arrestins, b-arrestin 1 and b-arrestin 2, are multifunctional signaling molecules15 with well established functions in the
desensitization and endocytosis of diverse cell surface receptors16–19.
They have been proposed to regulate the activities and/or subcellular
distributions of various signaling molecules, including the kinase Akt
(also called protein kinase B), Src family kinases and certain components of the mitogen-activated protein kinase family, such as
Erk15,20,21. In addition to the pivotal functions of b-arrestins in
signaling regulation in the cytoplasm, b-arrestin 1 has a function in
the nucleus, where it binds and recruits histone acetylase p300 to
specific gene promoters and increases local acetylation of histone H4
and gene transcription22. The b-arrestins are universally expressed, but
the neural and immune systems have much higher expression of
b-arrestin proteins23,24. It has been shown that b-arrestin 2 negatively
modulates Toll-like receptor signaling in innate immunity, but it has
1Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of
Sciences, Chinese Academy of Sciences, Shanghai 200031, China. 2Joint Immunology Laboratory of Institute of Health Sciences and Shanghai Institute of Immunology,
Shanghai JiaoTong University School of Medicine and Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200025, China. 3Institute of
Neurology, Huashan Hospital, Shanghai Medical College of Fudan University, Shanghai 200040, China. 4Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 200031, China. 5Department of Neurology, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200025, China. 6E-Institutes
of Shanghai Universities, Shanghai 200240, China. Correspondence should be addressed to G.P. ([email protected]) or J.Z. ([email protected]).
Received 29 March; accepted 15 June; published online 8 July 2007; doi:10.1038/ni1489
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102
*
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CD4
CD8
0
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72
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*
25
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**
**
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Arr1tg
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*
**
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CD4
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LN
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Figure 1 Involvement of b-arrestin 1 in the homeostasis and survival of CD4+ T cells. (a) Flow cytometry of the
expression of CD4 and CD8 in lymph node cells (LN) and splenocytes (Spleen) from Arrb1–/– mice, Arrb1tg mice
and wild-type mice (WT) at 13–18 weeks of age. Numbers in quadrants indicate percent CD4+CD8– cells (top left)
or CD4–CD8+ cells (bottom right). Below, cell numbers for various populations of splenocytes (left) and lymph node
cells (right). Data are representative of four experiments with more than ten mice per group. (b,c) Survival assays of
splenic CD4+ and CD8+ T cells from Arrb1–/–, Arrb1tg and wild-type mice left untreated (Naive; b) or activated with
anti-CD3 and anti-CD28 (c). Viable cells were identified by propidium iodide and annexin V exclusion. Data are
representative of three experiments. (d,e) Flow cytometry of total peripheral blood mononuclear cells from
Arrb1–/–, Arrb1tg and wild-type mice (five mice per group) immunized intraperitoneally with 100 mg staphylococcal
enterotoxin B (SEB) for the population expansion of T cell receptor Vb8.1,2+ (d) or Vb6+ (e) CD4+ and CD8+ T cells.
Values are relative to those at time 0. Data are pooled from three independent experiments. *, P o 0.05, and
**, P o 0.01, versus the wild-type control.
also been reported that b-arrestin 2 regulates the development of
allergic asthma25,26. The function of b-arrestin 1 in immune cells,
however, remains mostly uncharacterized.
Here we report that b-arrestin 1 has a positive regulatory function
in CD4+ T cell survival and homeostasis. This function of b-arrestin 1
in T cell biology seems to be mediated mainly by its nuclear function,
as it promoted acetylation of histone H4 at the Bcl2 locus and Bcl2
expression. The physiological relevance of CD4+ T cell regulation by
b-arrestin 1 was strongly supported by our findings that in mice
‘programmed’ to develop the autoimmune demyelinating disease
experimental autoimmune encephalomyelitis (EAE), the disease phenotype was alleviated or aggravated considerably in Arrb1-knockout
or Arrb1-transgenic mice, respectively, and that higher expression of
Arrb1 and Bcl2 in autoreactive CD4+ T cells from multiple sclerosis
patients contributed to the survival of these cells. Our results collectively identify b-arrestin 1–dependent mechanisms critical for the
regulation of CD4+ T cell physiology and link b-arrestin 1 to the
pathogenesis of autoimmunity.
RESULTS
Regulation of CD4+ T cell survival and homeostasis
Analysis of the immune system, in which the expression of b-arrestins
was relatively high (Supplementary Fig. 1a online), showed that adult
Arrb1–/– mice had fewer peripheral CD4+ cells, but not CD8+ T cells
and B cells, than wild-type mice had (Fig. 1a and Supplementary
Fig. 1b). Homeostasis of peripheral CD4+ T cells is maintained
between their production and their elimination. As our analysis of
thymocyte populations of Arrb1–/– and wild-type mice by flow
cytometry showed no significant differences (Supplementary Fig. 1c),
we investigated the effects of b-arrestin 1 on the survival of peripheral
CD4+ T cells. We purified CD4+ T cells from the spleens of
2
Arr1tg
** **
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10 15 20
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0.5
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Time after SEB injection (d)
Time after culture (h)
CD4 cells
(‘fold expansion’)
*
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2.0
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**
CD8 cells
(‘fold expansion’)
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© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
CD8
Spleen
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20.2
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100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
50
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(‘fold expansion’)
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25
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Live CD4 T cells (%)
8.1
30.4
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d
Activated T cells
Live CD8 T cells (%)
Spleen
CD4
10.3
10.7
101
c
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13.0
102
100
104 31.9
103
LN
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Arr1 –/–
WT
20.4
Live CD4 T cells (%)
Arr1tg
104 21.5
103
Live CD8 T cells (%)
a
Vβ6
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0.5
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5 10 15 20
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Time after SEB injection (d)
Arrb1–/– mice, Arrb1-transgenic (Arrb1tg) mice and wild-type mice
and activated some with antibody to CD3 (anti-CD3) and anti-CD28,
followed by culture in medium without additional cytokines. After
activation, CD4+ T cells from Arrb1tg mice showed increased survival,
whereas both naive and activated CD4+ T cells from Arrb1–/– mice
were more prone to apoptosis than those from the wild-type mice
(Fig. 1b,c). These data indicated that b-arrestin 1 promotes the
survival of naive and activated CD4+ T cells in vitro.
To examine the function of b-arrestin 1 in vivo, we challenged
Arrb1–/–, Arrb1tg and wild-type mice with staphylococcal enterotoxin
B and monitored the responding variable b-chain (Vb)8.1,2 and
nonresponding Vb6 T cells at various intervals11. Vb8.1,2 T cell
populations in mice of the various genotypes expanded similarly on
day 3 after injection of staphylococcal enterotoxin B, but their viability
decreased thereafter (Fig. 1d). During the subsequent observation
period, it was evident that Arrb1tg mice had more circulating CD4+
Vb8.1,2 T cells than wild-type control mice had, whereas Arrb1–/– mice
had fewer such cells than wild-type control mice had (Fig. 1d),
suggesting that b-arrestin 1 confers substantial protection to the
responding CD4+ T cells from staphylococcal enterotoxin B–induced
cell death in vivo. The protective effect seemed to be specific to CD4+
T cells, as attrition of the corresponding CD8+ T cells was similar in all
three groups of mice (Fig. 1d). In contrast, the populations of
nonresponding Vb6 T cells (CD8+ or CD4+) were not affected in
these mice throughout the observation period (Fig. 1e).
It is well known that the cytokine interleukin 2 (IL-2) is essential for
CD4+ T cell survival27–29 and that after activation in vitro, CD4+
T cells make large amounts of IL-2, which prevents apoptosis1. We
found, however, that IL-2 production was slightly higher in CD4+
T cells from Arrb1–/– mice than in cells from wild-type mice after
activation with anti-CD3 and anti-CD28 (Supplementary Fig. 2a
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a
Time (h) 0 48 96 168
0 12 24 36 48 60 72 84
40 44 48 52 56 60 64
Actin
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© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
**
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Figure 2 Promotion of Bcl2 expression by b-arrestin 1 in CD4+ T cells.
(a) Immunoblot of the expression of b-arrestin 1 (Arrb1), b-arrestin 2
(Arrb2), Bcl-2 and Bax in splenic CD4+ T cells activated with anti-CD3
and anti-CD28 (time, above lanes). b-actin (Actin), loading control.
(b–d) Quantitative RT-PCR analysis of Bcl2 and Bax mRNA (left) and
immunoblot analysis of b-arrestin 1, Bcl-2 and Bax (right) in wild-type,
Arrb1–/– (b), Arrb1tg (c) and Arrb2–/– (d) CD4+ T splenocytes activated
with anti-CD3 and anti-CD28 (time, below graphs and above lanes).
Results for quantitative RT-PCR are normalized to those obtained for the
gene encoding mouse hypoxanthine phosphoribosyl transferase and are
presented relative to those obtained at time 0, set as 100%. b-actin,
protein loading control (immunoblot). **, P o 0.01, versus the
corresponding control. Quantitative RT-PCR data are pooled from three
independent experiments; all immunoblot analyses are representative of
at least three independent experiments.
**
–/–
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online). Thus, the lower survival of CD4+ T cells from Arrb1–/– mice
did not seem to be due to lower production of IL-2.
Epigenetic regulation of Bcl2 in CD4+ T cells
We further investigated the molecular mechanisms underlying
b-arrestin 1–induced survival of CD4+ T cells. Given the known
functions of the Akt30 and Erk31 signaling pathways in T cell survival
and the positive effect of b-arrestin 1 on the activation of Akt and
Erk15, we investigated whether b-arrestin 1 enhanced CD4+ T cell
survival by increasing Akt and Erk activity. We activated CD4+ T cells
purified from Arrb1–/– and wild-type mice as described above or left
them untreated and assessed activation of Akt and Erk by monitoring
their phosphorylation status. We found that b-arrestin 1 had no effect
on Erk phosphorylation in naive or activated CD4+ T cells and that
Akt activation was slightly lower in Arrb1–/– CD4+ T cells only after
their activation (Supplementary Fig. 2b).
Given that b-arrestin 1 promoted CD4+ T cell survival in both naive
and activated states (Fig. 1) and that b-arrestin 1 did not seem to
affect the activation of Akt and Erk in naive cells, we reasoned that
there must be additional mechanisms by which b-arrestin 1 regulates
T cell survival. We thus did microarray analyses with Affymetrix gene
chips and found that inhibition of Arrb1 expression mediated by
Arrb1-specific small interfering RNA (siRNA) downregulated transcription of the antiapoptotic gene Bcl2 but did not affect other Bcl2
family members, such as Bax, Bcl2l11 (encoding Bim) and Bcl2l1
(encoding Bcl-xL; data not shown). We confirmed this finding in the
Jurkat cell line by reverse transcription and quantitative real-time PCR
and further found that it was nuclear b-arrestin 1 that upregulated the
transcription of Bcl2, as both b-arrestin 2 and mutant b-arrestin 1
with a Q394L substitution, located exclusively in the cytoplasm32,33,
had no such effect (Supplementary Fig. 3b online).
Given that Bcl-2 is critically involved in regulating the apoptosis of
both naive and activated CD4+ T cells1,11 and given the potential
regulation of its expression by b-arrestin 1, we examined the expression
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of Arrb1 and Bcl2 in CD4+ T cells after activation. We found that in the
first 2 d after CD4+ T cell activation, b-arrestin 1 and Bcl-2 protein
gradually decreased but then mostly recovered by day 3 and remained
high thereafter (Fig. 2a). To analyze the changes of Arrb1 and Bcl2
expression in more detail, we collected samples every 4 h from 40 h to
64 h after CD4+ T cell activation and found that the initial recovery of
b-arrestin 1 seemed to precede that of Bcl-2 (Fig. 2a, right). To investigate whether b-arrestin 1 regulates Bcl2 expression in these cells, we
obtained CD4+ T cells from Arrb1–/– and wild-type mice, activated
them in vitro and assessed Bcl2 mRNA and Bcl-2 protein at various
intervals thereafter. As noted above, Bcl2 showed a dynamic pattern of
regulation after activation of wild-type CD4+ T cells, with a gradual and
significant decrease during the first 2 d and recovery to original
expression by day 3 (Fig. 2b). Arrb1–/– CD4+ T cells had significantly
less Bcl2 mRNA than wild-type cells did, even at time zero, indicating
that b-arrestin 1 is required for normal Bcl2 expression in physiological
conditions (Fig. 2b). Moreover, the dynamic pattern of Bcl2 expression
after activation of wild-type CD4+ T cells was completely abolished in
Arrb1–/– cells (Fig. 2b), indicating that b-arrestin 1 is critically involved
in the regulation of Bcl2 after CD4+ T cell activation. Indeed, the
amount of Bcl2 mRNA in Arrb1–/– cells was similar to the lowest noted
during activation of wild-type CD4+ T cells (Fig. 2b).
We further examined CD4+ T cells obtained from Arrb1tg mice and
found that b-arrestin 1 protein recovered faster in Arrb1tg CD4+
T cells after activation, such that at 48 h after activation the cells
contained significantly more b-arrestin 1 than wild-type cells did
(Fig. 2c, right). Notably, at this time point, Bcl2 mRNA and Bcl-2
protein were also higher in Arrb1tg CD4+ T cells than in cells
from wild-type mice (Fig. 2c), consistent with the critical function
of b-arrestin 1 in the regulation of Bcl2 expression. Additionally, as
b-arrestin 2 protein changed in a way similar to b-arrestin 1 during
activation of these cells, we also investigated the expression of Bcl2 in
CD4+ T cells from Arrb2–/– mice and found that its expression pattern
was normal (Fig. 2d). These data indicate b-arrestin 1 but not
b-arrestin 2 specifically regulates Bcl2 expression. The critical function
of b-arrestin 1 in the positive regulation of Bcl2 expression probably
accounts for the lower survival of Arrb1–/– CD4+ T cells, as the
apoptosis features in these cells were very similar to those of Bcl2knockout CD4+ T cells13.
We used Jurkat T cells to investigate whether b-arrestin 1 promotes
Bcl2 expression through direct regulation of its transcription. We
found that transient transfection of Arrb1-specific siRNA or the Arrb1
gene significantly decreased or increased, respectively, Bcl2 mRNA and
Bcl-2 protein (Supplementary Fig. 4a online), consistent with the
results reported above (Supplementary Fig. 3b). Furthermore, the
increased Bcl-2 protein caused by overexpression of Arrb1 could be
3
Bcl2
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H4Ac
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Epigenetic regulation of Bcl2 expression by p300
Histone acetylation is regulated by the activities of histone
acetyltransferase and histone deacetylase. Studies have shown that
0
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u2
.
u2 5 ×
. 1
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. 1
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81 10
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×
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Histone acetylation ratio
WT
blocked by either the mRNA transcription inhibitor actinomycin D or
the protein synthesis inhibitor cycloheximide (Supplementary
Fig. 4b,c). These data are consistent with the idea that b-arrestin 1
promotes Bcl2 expression by increasing its transcription. CREB34 and
NF-kB35 are transcription factors known to regulate Bcl2 expression.
However, overexpression of Arrb1 did not cause any significant
increase in CREB- or NF-kB-mediated transcriptional activity, as
assayed by reporter plasmids (Supplementary Fig. 4d,e). Therefore,
it seems unlikely that b-arrestin 1 regulates Bcl2 expression through
direct activation of these two transcription factors.
Another mechanism by which b-arrestin 1 could promote gene
expression is by increasing local acetylation of histone H4 at target
promoter regions22. Acetylation of histone H4 at the Bcl2 promoter
changed dynamically after CD4+ T cell activation (Fig. 3a), which
paralleled the changes of Bcl2 expression (Fig. 2). For example,
acetylation of histone H4 at the Bcl2 promoter region gradually
reached its lowest point at approximately 48 h after CD4+ T cell
activation and then recovered to its normal amount at 72 h. As a
control, acetylation of histone H3 at the Bcl2 promoter was
unchanged. Consistent with our earlier results that Bcl2 expression
was much lower in Arrb1–/– CD4+ T cells and remained low after T cell
activation, we noted a similar pattern for acetylation of histone H4 at
the Bcl2 promoter in Arrb1–/– CD4+ T cells, as it remained similar to
the lowest acetylation found in wild-type CD4+ T cells during their
activation (Fig. 3b). However, acetylation of histone H4 at the Bcl2
promoter was significantly higher at 48 h after activation in Arrb1tg
CD4+ T cells than in wild-type cells (Fig. 3c), a result reminiscent of
the faster recovery of expression of Arrb1 and Bcl2 at the same time
point in these cells (Fig. 2). Further, b-arrestin 1 promoted acetylation
of histone H4 in a region approximately spanning from 15,000 base
pairs upstream to 186,000 base pairs downstream of the Bcl2 transcription start site (Fig. 3d). These data indicate that b-arrestin 1 is
critically involved in promoting acetylation of histone H4 at the Bcl2
locus, which is most probably the mechanism of upregulation of Bcl2
expression by b-arrestin 1.
4
b
150
Relative H4Ac
150
Relative H3Ac
a
Relative H4Ac
Figure 3 Promotion of acetylation of histone H4
at the Bcl2 locus by b-arrestin 1. Chromatinimmunoprecipitation analysis of splenic CD4+
T cells activated with anti-CD3 and anti-CD28.
(a) Analysis of acetylated histone H4 (H4Ac; left)
and acetylated histone H3 (H3Ac; right) in wildtype mice. (b,c) Analysis of acetylated histone
H4 in Arrb1–/– and wild-type mice (b) and in
Arrb1tg and wild-type mice (c). Values are
relative to the acetylation at time 0, set as
100%. (d) Chromatin immunoprecipitation and
quantitative PCR analysis of the acetylation of
histones H4 and H3 at and surrounding the
Bcl2 locus in Arrb1–/– and wild-type mice.
Data represent the ratio of acetylation at a
specific position of the Bcl2 locus (horizontal
axis) in wild-type CD4+ T cells to that in
Arrb1–/– CD4+ T cells and are normalized to
the corresponding input controls. 0, start
point of transcription; u, upstream of the start
point. **, P o 0.01, versus the corresponding
control. All data are pooled from three
independent experiments.
Relative H4Ac
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Distance (base pairs)
b-arrestin 1 can be recruited to promoters of specific target genes and
promote local acetylation of histone H4 by specifically binding to p300
(ref. 22), a histone acetyltransferase36. Thus, we investigated the
function of p300 in b-arrestin 1–mediated acetylation of histone H4
at the Bcl2 promoter and as a means of regulating Bcl2 transcription.
Consistent with the specific function for b-arrestin 1 in the
regulation of Bcl2 transcription, we found that b-arrestin 1 but not
b-arrestin 2 or the b-arrestin 1 cytoplasmic mutant (Q394L) increased
acetylation of histone H4 of the Bcl2 promoter region (Supplementary Fig. 5 online). Furthermore, acetylation of histone H4 at the Bcl2
promoter and Bcl2 transcription were stimulated considerably by
overexpression of p300 in Jurkat cells. These effects of p300 were
augmented by coexpression of b-arrestin 1 and were blocked by
coexpression of Arrb1-specific siRNA (Supplementary Fig. 5c,e).
Moreover, a dominant negative mutant of p300 strongly attenuated
the stimulatory effect of b-arrestin 1 on both acetylation of histone H4
at the Bcl2 promoter region and Bcl2 transcription (Supplementary
Fig. 5c,e). These data indicate that p300 is critical in b-arrestin
1–mediated hyperacetylation of histone H4 at the Bcl2 promoter
region and in Bcl2 transcription.
Autoimmune demyelinating disease and b-arrestin 1
Regulation of CD4+ T cell apoptosis by cell death–signaling molecules
is critical for regulating the frequency and activity of autoreactive
CD4+ T cells; disturbance of these cells may contribute to inflammatory pathologies in autoimmune conditions. To assess the pathophysiological relevance of b-arrestin 1 function in CD4+ T cells, we used
the EAE mouse model37 to investigate whether b-arrestin 1 regulates
the pathogenesis of autoimmune disease. We induced EAE in Arrb1–/–,
Arrb1tg and wild-type mice (all on the same C57BL/6 background) by
injecting a peptide of rat myelin oligodendroglial glycoprotein (MOG)
amino acids 35–55. EAE disease onset was delayed significantly in
Arrb1–/– mice (wild-type, 9.7 ± 2.9 d; Arrb1–/–, 16.3 ± 3.0 d) and was
accompanied by significantly lower clinical scores (maximum clinical
score: wild-type, 3.2 ± 0.2; Arrb1–/–, 1.4 ± 0.4; Fig. 4a). In contrast,
although there was no significant difference in the timing of disease
onset (wild-type, 9.7 ± 2.9 d; Arrb1tg: 8.7 ± 0.8 d), Arrb1tg mice had
greater clinical severity (maximum clinical score: Arrb1tg, 4.2 ± 0.3;
wild-type, 3.2 ± 0.2). The differences between the experimental groups
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Arr1tg
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H incorporation
(c.p.m. × 102)
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Time (h) 0
MOG CD4 T cells MOG CD8 T cells
were highly significant by the linear regression method38 and by twoway analysis of variance (P o 0.001; Fig. 4a).
In spinal cord sections, we found a notable paucity of inflammatory
lesions and demyelination in Arrb1–/– tissues and more severe inflammatory lesions and demyelination in Arrb1tg tissues than in wild-type
tissues (Fig. 4b). Given that b-arrestin 1 expression in CD4+ T cells
was critically involved in regulating the apoptosis of these cells, we
sought to determine whether dysregulated apoptosis might contribute
to the effects of b-arrestin 1 on the disease progression and severity of
EAE. We obtained splenocytes from Arrb1–/–, Arrb1tg and wild-type
mice in which EAE had been induced, stimulated the cells with MOG
peptide and evaluated their proliferation by [3H]thymidine incorporation assay. MOG stimulation greatly increased the proliferation of
Arrb1tg splenocytes to more than double the proliferation of wild-type
cells, but the MOG-stimulated proliferation was significantly attenuated in Arrb1–/– cells (Fig. 4c). We also tested the viability of MOGspecific T cells isolated from mice with EAE. Consistent with the
results reported above, MOG-specific CD4+ T cells from Arrb1tg mice
had a significantly higher survival rate, whereas those from Arrb1–/–
b
350
300
**
Control
MS
250
200
150
100
50
0
c
250
Relative BCL2 mRNA
a
Relative ARRB1 mRNA
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
Arr1tg
10
3
WT
WT
**
15
200
*
Figure 4 Critical involvement of b-arrestin 1 in EAE. (a,b) Induction
of EAE in Arrb1–/–, Arrb1tg and wild-type mice. (a) Clinical scores of mice
monitored daily for signs of disease, assigned according to disease severity.
Left, each line represents the average scores of 10 Arrb1–/– mice,
10 Arrb1tg mice and 14 wild-type mice. Right, linear regression analysis
of the data at left. P o 0.001. Data are pooled from experiments done
four times. (b) Sections of spinal cords from mice 20 d after EAE induction;
sections were fixed and were stained with hematoxylin and eosin (H&E) to
demonstrate the degree of inflammation and with Luxol fast blue to show
the degree of demyelination. Original magnification, 200 (Luxol fast blue)
or 100 (hematoxylin and eosin). Images are representative of three to four
mice per group. (c) Proliferation assay of splenocytes from Arrb1tg, Arrb1–/–
and wild-type mice maintained for 8 d after EAE induction, assessed as
[3H]thymidine incorporation with or without (No stim) restimulation with
MOG peptide. **, P o 0.01, versus the wild-type control. Data are pooled
from three independent experiments. (d) Viability of CD4+ and CD8+ T cells
purified after MOG restimulation of splenocytes isolated from mice
maintained for 8 d after EAE induction; cells were then cultured in medium
and assessed by propidium iodide and annexin V exclusion. *, P o 0.05,
and **, P o 0.01, versus the wild-type control. Data are pooled from three
independent experiments.
Control
MS
Clone 1
Control Arrb1si
150
Actin
100
Arrb1
Arrb2
50
Bcl-2
mice were more susceptible to apoptosis (Fig. 4d). In addition, we
analyzed the percentage of CD62L+, CD44+ and CD4+CD25+Foxp3+
regulatory T cells in peripheral CD4+ T cell populations of both naive
mice (weeks 7–8 and 15–16) and mice with EAE (weeks 7–8). We
found no significant differences among the three groups (wild-type,
Arrb1–/– and Arrb1tg), indicating that the proportion of naive,
memory and regulatory T cell subsets of CD4+ T cells is not selectively
affected by b-arrestin 1 (Supplementary Fig. 6 online). These data
suggest that b-arrestin 1 has an important function in the induction
and severity of EAE by altering the survival and functional properties
of MOG-specific CD4+ T cells.
The expression of b-arrestin 1 in splenocytes is upregulated when
mice are induced to develop EAE39. We further confirmed that
b-arrestin 1 expression in CD4+ T cells was much higher after EAE
induction (Supplementary Fig. 7 online). Given the positive function
of b-arrestin 1 in promoting CD4+ T cell survival and the finding that
its expression was much higher after EAE induction, we sought to
determine whether and to what extent b-arrestin 1 expression affects
the functional properties of CD4+ T cells and myelin-reactive T cells in
patients with multiple sclerosis.
We purified CD4+ T cells from samples of peripheral blood
mononuclear cells obtained from 14 patients with relapsing-remitting
multiple sclerosis (12 female and 2 male; all 21–55 years of age) and
Clone 2
Control Arrb1si
d
Clone 1
Clone 2
100
75
*
50
**
25
0
Time (h)
0
24
48
Live CD4 T cells (%)
WT
4
Live CD4 T cells (%)
5
Clinical score
Clinical score
a
Control
Arrb1si
100
75
*
50
**
25
0
Time (h)
0
24
48
0
Figure 5 Critical involvement of b-arrestin 1 in the pathogenesis of multiple sclerosis. (a,b) Quantitative RT-PCR analysis of ARRB1 mRNA (a) and
BCL2 mRNA (b) in CD4+ T cells purified from samples of peripheral blood mononuclear cells obtained from 14 patients with relapsing-remitting multiple
sclerosis (MS) and 14 healthy controls. Values are relative to control values, set as 100%. *, P o 0.05, and **, P o 0.01, versus the healthy control.
(c) Immunoblot analysis of b-arrestin 1 and Bcl-2 in lysates of MBP-specific CD4+ T cell clones from patients with multiple sclerosis; clones were infected
with lentivirus carrying ARRB1-specific siRNA (Arrb1si) or empty vector (Control). Actin, loading control. Data are representative of three independent
experiments. (d) Viability of MBP-specific CD4+ T cells infected with lentivirus expressing ARRB1-specific siRNA or empty vector and then cultured
(time, horizontal axes). Viable cells with green fluorescent protein fluorescence were identified by propidium iodide exclusion. *, P o 0.05, and
**, P o 0.01, versus the empty vector control. Data are pooled from three independent experiments.
NATURE IMMUNOLOGY
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5
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES
14 gender- and age-matched healthy controls. We used quantitative
RT-PCR to evaluate the transcription of ARRB1 and BCL2 in these
cells. We detected significantly higher transcription of ARRB1 and
BCL2 in CD4+ T cells from patients with multiple sclerosis than in
those from healthy people (Fig. 5a,b). In addition, when we analyzed
six independent T cell clones reactive to myelin basic protein (MBP),
we found that clones derived from patients with multiple sclerosis had
higher expression of ARRB1 and BCL2 than did clones generated from
healthy people (data not shown). Furthermore, when we ‘knocked
down’ b-arrestin 1 expression in MBP-specific CD4+ T cell clones
generated from patients with multiple sclerosis40 by a lentivirusmediated RNA-mediated interference approach, we found much less
expression of Bcl-2 (Fig. 5c). MBP-specific CD4+ T cells infected with
lentivirus carrying ARRB1-specific siRNA were much more susceptible
to apoptosis than were cells infected with a control lentivirus (Fig. 5d).
These data collectively indicate that CD4+ T cells specifically involved
in multiple sclerosis have higher expression b-arrestin 1, which may
contribute to the altered survival and aberrant functional properties of
these inflammatory T cells in multiple sclerosis.
DISCUSSION
The immune system is a very dynamic system in mammals, and cell
apoptosis in this system is critically regulated by various intracellular
and extracellular signaling mechanisms. Our results here have demonstrated that b-arrestin 1 specifically regulates the survival and homeostasis of CD4+ T cell and thus affects adaptive immune responses.
Although we found that impaired Akt activation in activated Arrb1–/–
CD4+ T cells might contribute to the impaired survival of these
activated cells, the survival function of b-arrestin 1 may instead be
attributed mainly to its nuclear function through epigenetic regulation
of Bcl2 expression in both naive and activated CD4+ T cells. We also
found that b-arrestin 1 is critically involved in the induction and
severity of autoimmune demyelinating disease and streptozotocininduced type 1 diabetes (data not shown). In the autoimmune
demyelinating disease, the encephalitogenic CD4+ T cells had higher
expression of b-arrestin 1 that seemed to promote the survival of these
cells. These results suggest a critical function for b-arrestin 1 in CD4+
T cell survival and autoimmunity regulation.
Our mechanistic analyses showed that b-arrestin 1 epigenetically
regulates Bcl2 expression in CD4+ T cells. Bcl-2 accomplishes important
functions in cell survival and death by controlling the integrity of
mitochondrial membranes and the release of proapoptotic molecules
residing in the mitochondria7. In T cells, Bcl2 expression thus should be
critically controlled; otherwise, T cell homeostasis and immune
responses will probably be disturbed. The regulation mechanism for
Bcl2 expression, however, remains mostly unknown. Here we found that
b-arrestin 1 promoted acetylation of histone H4 at the Bcl2 locus and
Bcl2 expression in both naive and activated CD4+ T cells. We also found
that this mechanism is conserved between mice and humans. Thus, in
addition to demonstrating a previously unknown mechanism regulating
CD4+ T cell survival, our results also provide an epigenetic view of
Bcl2 regulation, which has not explored before to our knowledge.
We noted involvement of b-arrestin 1 in T cell survival and
homeostasis mainly for peripheral CD4+ but not CD8+ T cells and
thymocytes. In thymocytes, b-arrestin 1 expression was very low
compared with that of splenocytes or lymphocytes, which could
explain why b-arrestin 1 had little effect on the thymocytes. Although
there was no substantial difference in b-arrestin 1 protein in peripheral
CD4+ and CD8+ T cells (data not shown), the effects of b-arrestin 1
on CD4+ and CD8+ T cell survival were very different. CD4+ T cells
deficient in b-arrestin 1 had much lower survival than wild-type cells
6
had, whereas Arrb1–/– CD8+ T cells had no notable defect in survival.
Mechanistic analysis showed that b-arrestin 1 located in the nucleus
upregulated acetylation of histone H4 of the Bcl2 promoter and
thus Bcl2 expression; overexpression of a cytoplasmic mutant of
b-arrestin 1 (Q394L) did not have any effect on Bcl2 expression. We
also examined whether the subcellular distribution of b-arrestin 1 in
CD4+ and CD8+ T cells is different. We found that for both naive and
activated cells, CD4+ T cells had much more nuclear b-arrestin 1 than
CD8+ T cells had (data not shown). Thus, the difference in the
subcellular distribution of b-arrestin 1 in CD4+ and CD8+ T cells
might account for the differential effect of b-arrestin 1 on the survival
of CD4+ and CD8+ T cells.
Activation of G protein–coupled receptors recruits both b-arrestin 1
and b-arrestin 2 to the cell membrane, and subsequent interactions
between phosphorylated G protein–coupled receptors and the
b-arrestins induce receptor endocytosis and signal attenuation. However, accumulating evidence also indicates possible functional differences between the two b-arrestin subtypes as well as their distinct
receptor specificity41. Here we have shown that b-arrestin 1 but not
b-arrestin 2 specifically promoted acetylation of histone H4 of the Bcl2
locus and Bcl2 expression. This difference between b-arrestin 1 and
b-arrestin 2 is consistent with published results22 and is presumably
due to their different subcellular distributions. Their functional differences notwithstanding, both b-arrestin 1 and b-arrestin 2 seem to
regulate immune responses; our results here have shown that b-arrestin
1 promoted CD4+ T cell survival and autoimmune conditions, whereas
another published study has reported that b-arrestin 2–deficient mice
have greatly reduced clinical symptoms of asthma mainly because of
the effect of b-arrestin 2 on immune cell migration25. It thus seems
that, collectively, b-arrestins have positive functions in adaptive
immune responses, albeit through different mechanisms.
It has been shown that b-arrestin 1 is directly involved in the
regulation of gene expression and that this effect can be enhanced by
signals from G protein–coupled receptors22. Expression of the delta
opioid receptor has been detected after CD4+ T cells are activated
in vitro42. Notably, we have found that additional activation of delta
opioid receptor induced Bcl2 expression in a b-arrestin 1–dependent
way (unpublished data). These data characterize b-arrestin 1 as an
important signaling regulator that may directly relay environmental
signals to influence CD4+ T cell survival, thus affecting the adaptive
immune response. That function, considered in addition to the
intrinsic function of b-arrestin 1 in CD4+ T cells, indicates important
mechanisms by which the immune system may be modulated by
b-arrestin 1 in physiological conditions.
Survival of CD4+ T cells is directly linked to the number and
function of CD4+ T cells, known to be important in immune system
homeostasis and autoimmune conditions. We have demonstrated that
CD4+ T cells and MBP-reactive T cells of patients with multiple
sclerosis had higher expression of b-arrestin 1 than those of healthy
people. Overexpression of ARRB1 in CD4+ T cells may be associated
with hyperactivity of CD4+ T cells as one of the immunological
features of multiple sclerosis43, although this awaits further investigation. Nevertheless, one therapeutic strategy for the treatment of
multiple sclerosis and some other human autoimmune diseases is to
deplete or reduce the number and function of CD4+ T cells. Clinical
trials have indicated that therapeutic reduction or depletion of CD4+
T cells by monoclonal antibodies may favorably alter the clinical
course of multiple sclerosis, although the effect is not statistically
significant44,45. It may thus be prudent to further evaluate the function
of b-arrestin 1 in therapeutic strategies targeting CD4+ T cells for the
treatment of autoimmune diseases such as multiple sclerosis.
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METHODS
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
Study subjects. Subjects volunteering for this study were patients from the
outpatient clinic of Huashan Hospital of Fudan University and Ruijin Hospital
of JiaoTong University who were diagnosed with clinically defined relapsingremitting multiple sclerosis. Blood samples were obtained from subjects after
informed-consent procedures were completed in accordance with the guidelines of local institutional review boards. All healthy subjects for this study were
volunteers from the Shanghai Blood Center and personnel of the Institute for
Biochemistry and Cell Biology; all provided informed consent.
Mice, cell lines, tranfection and lentivirus infection. C57BL/6 mice were from
the Shanghai Laboratory Animal Center (Chinese Academy of Sciences).
Arrb1–/– and Arrb2–/– mice were on a C57BL/6 background. Arrb1tg mice were
generated in the laboratory of G.P.; expression of hemagglutinin-tagged human
Arrb1 was under control of the human cytomegalovirus promoter (L.Z.,
R. Yang, J. Chai and G.P., unpublished data). Arrb1tg mice used here were
backcrossed onto the C57/BL6 background for more than nine generations and
did not manifest autoimmune conditions, as determined by autoantibody titers
and assessment of clinically overt disease. The animal protocol was approved by
the institutional animal use committee of the Shanghai Institutes for Biological
Sciences (Chinese Academy of Sciences). All mice were maintained in
pathogen-free conditions and were genotyped before use.
Jurkat and HEK293 cells (American Type Culture Collection) were maintained
in RPMI 1640 medium and MEM (Gibco-BRL), respectively. Jurkat cells were
transfected with Nucleofector (Amaxa). By calcium phosphate precipitation,
HEK293 cells were cotransfected with pNF-kB-Luc or pCREB-TA-Luc, pRL-TK
(Clontech) and other plasmids. At 36 h after transfection, luciferase activity was
measured with the Dual Luciferase Reporter Assay system normalized to control
luciferase activity. Cells treated with forskolin (10 ng/ml; Sigma) or recombinant
human tumor necrosis factor (10 ng/ml; PeproTech) served as positive controls.
Antibodies, reagents, plasmids and siRNA. Mouse anti-Bcl-2 (7; 610539)
and anti-Bax (3; 610983), phycoerythrin-conjugated antibodies to mouse CD4
(RM4-5; 553048), CD8 (53-6.7; 553032), CD44 (IM7; 553134), B220 (RA3-6B2;
553089) and Vb6 (RR4-7; 553194), and fluorescein isothiocyanate–conjugated
antibodies to mouse CD4 (RM4-5; 01064D), CD8 (53-6.7; 553030), CD3 (1452C11; 553061), CD25 (7D4; 553072) and Vb8.1,2 (MR5-2; 553185) were from BD
Biosciences. Allophycocyanin-conjugated anti–mouse CD62L (MEL-14-H2.100;
130-091-805) was from Miltenyi Biotec; the allophycocyanin-conjugated antimouse, rat Foxp3 Staining Set was from eBioscience; antibodies to acetylated
histone H4 (06-866) and to acetylated histone H3 (06-599) were from Upstate
Biotechnology; IRDye 800CW–conjugated, affinity-purified anti-mouse
IgG (610-131-121) and anti-rabbit IgG (600-431-384) were from Rockland;
anti-b-actin (1A4; A2547), actinomycin D and dexamethasone were from Sigma;
anti-Erk (9122) and antibody to Erk phosphorylated at Ser217 and Ser221
(9121L) were from Cell Signaling; and anti-Akt (KC-5A05) and antibody to Akt
phosphorylated at Ser473 (KC-5A04) were from Kangchen. Staphylococcal
enterotoxin B was from Biotinge Biomedicine. Plasmids expressing cytomegalovirus b-galactosidase and hemagglutinin-tagged b-arrestin 1 (long form24),
b-arrestin 2 and b-arrestin 1 mutant (Q394L) were generated as described33,46.
Construction of the pBS-U6-Arrb1, pBS-U6-Arrb2 and pBS-U6-nonspecific
siRNA plasmids has been described33,47. Plasmids expressing wild-type p300
and a dominant negative mutant of p300 (deletion of the first cysteine- or
histidine-rich region) were from Upstate Biotechnology. Human MBP peptides
were synthesized and purified by the Peptide Core Laboratory of M.D. Anderson
Cancer Center; peptide purity was over 90%.
Cell purification, activation and culture. Splenic cells stained with purified rat
anti–mouse CD4 (RM4-5; 553043) or purified rat anti–mouse CD8a (53-6.7;
553027; both from BD Biosciences) were purified with goat anti–rat IgG
microbeads, separation columns and an AutoMACS sorter (Miltenyi Biotec),
yielding a purity of over 95% by flow cytometry. Purified CD4+ T cells were
activated with plate-bound purified hamster monoclonal anti–mouse CD3e
(CD3 e-chain; 5 mg/ml; 145-2C11; 553057) and hamster monoclonal anti-mouse
CD28 (4 mg/ml; 37.51; 553294; both from BD Biosciences) in RPMI 1640
medium supplemented with 2 mM L-glutamine, 5 mM b-mercaptoethanol,
100 U/ml of penicillin and 10% (vol/vol) FCS. MBP-specific CD4+ T cells were
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maintained in RPMI 1640 medium with MBP peptides48 (20 mg/ml) and
recombinant human IL-2 (100 U/ml; PeproTech). For analysis of cell survival,
T cells were cultured in RPMI 1640 medium without additional cytokines. At
various times, percent viable cells was determined by propidium iodide and
annexin V exclusion, or by propidium iodide exclusion only for green fluorescent protein–positive cells, with an Annexin V FLUOS Staining kit (Roche
Molecular Biochemicals). T cells from human blood samples were labeled with
purified mouse anti–human CD4 (S3.5; MHCD0400; Caltag) and were purified
with goat anti–mouse IgG microbeads, separation columns and an AutoMACS
sorter (Miltenyi Biotec), yielding a purity of over 90% by flow cytometry.
Flow cytometry. Cells were resuspended in PBS containing 1% (wt/vol) BSA
(Sigma-Aldrich). For surface staining of CD4, CD8, CD3, B220, T cell receptor
Vb8.1,2 and Vb6, cells were incubated for 1 h on ice with fluorochromeconjugated antibodies to various cell surface markers at the recommended
dilutions for isotype control antibodies. Stained cells were washed and were
analyzed with a FACSAria (BD Biosciences).
Reverse transcription and quantitative real-time PCR. Total RNA was
extracted from cultured cells with TRIzol (Invitrogen) according to the
manufacturer’s instructions. Oligo(dT) priming and superscript III reverse
transcriptase (Invitrogen) were used for reverse transcription of purified RNA.
All gene transcripts were quantified by quantitative PCR with Brilliant SYBR
Green QPCR Master Mix and a Light Cycler apparatus (Stratagene). Primer
pairs are listed in Supplementary Methods online.
Induction and evaluation of EAE. The encephalitogenic peptide of MOG
corresponding to residues 35–55 (BioAsia Biotechnology) used to induce EAE
had a purity of 95%. Acute EAE was induced by a subcutaneous immunization
with 300 mg of the MOG peptide in complete Fruend’s adjuvant containing heatkilled Mycobacterium tuberculosis (H37Ra strain; 5 mg/ml; BD Diagnostics).
Pertussis toxin (200 ng/mouse; List Biological Laboratories) in PBS was
administered intravenously on the day of immunization and 48 h later. Mice
6–8 weeks of age were weighed and examined daily for disease symptoms; they
were assigned scores for disease severity with the following EAE scoring scale: 0,
no clinical signs; 1, limp tail; 2, paraparesis (weakness, incomplete paralysis of
one or two hindlimbs); 3, paraplegia (complete paralysis of two hindlimbs); 4,
paraplegia with forelimb weakness or paralysis; and 5, moribund state or death.
Tissues for histological analysis were removed from mice 20 d after immunization and were immediately fixed in 4% (wt/vol) paraformaldehyde. Paraffinembedded sections of spinal cord 5–10 mm in thickness were stained with Luxol
fast blue or with hematoxylin and eosin and were examined by light microscopy.
The degree of demyelination and inflammatory infiltration was quantified
according to a published procedure49 on an average of three transverse sections
of spinal cord per mouse for a total of three to four mice per group.
Chromatin immunoprecipitation and immunoblot. Chromatin-immunoprecipitation assays were done according to the manufacturer’s instructions
(Upstate Biology). The presence of the target gene promoter sequences in both
the input DNA and the recovered DNA immunocomplexes was detected by
quantitative PCR. Data were normalized to those obtained with the corresponding DNA input control. The primer pairs for specific promoter regions were
from –1,000 base pairs to approximately +500 base pairs of the transcription
start site of the gene. Primer pairs are listed in Supplementary Methods.
For immunoblot analysis, protein bands were visualized by enhanced chemiluminescence. In some experiments, blots were incubated with IRDye 800CW–
conjugated secondary antibody (Rockland) and infrared fluorescence images
were obtained with the Odyssey infrared imaging system (Li-Cor Bioscience).
Proliferation assays and cytokine measurement. In proliferation assays, mouse
splenocytes (5 105 per well) were cultured in triplicate in complete DMEM in
96-well flat-bottomed plates. Cells were cultured for 72 h in the presence or
absence of MOG peptide (5 mg/ml). Cells were pulsed with 1 mCi [3H]thymidine
during the final 16–18 h of culture before being collected. Incorporation of
[3H]thymidine was measured as c.p.m. with a b-plate counter. For cytokine
measurement, CD4+ T cells (2 105 cells/well) were stimulated for 24 h in
96-well plates with anti-CD3 and anti-CD28 (both from BD Biosciences)
as described above (Cell purification, activation and culture). Supernatants
were collected for the measurement of IL-2 production by enzyme-linked
7
ARTICLES
immunoassay according to the manufacturer’s instructions (Pierce). A standard
curve was generated with known amounts of purified recombinant mouse IL-2.
Statistical analysis. Quantitative data are expressed as mean ± s.e.m. Statistical
significance was determined by one-way analysis of variance followed by
the Bonferroni post-hoc test for multiple comparisons or the two-tailed
Student’s t-test. A P value of less than 0.05 was considered statistically
significant. The linear regression method38 and two-way analysis of variance
were used for EAE experiments.
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
Note: Supplementary information is available on the Nature Immunology website.
ACKNOWLEDGMENTS
We thank R.J. Lefkowitz (Duke University Medical Center) for rabbit polyclonal
anti-b-arrestin (A1CT) and for Arrb1–/– and Arrb2–/– mice; Y. Shi,
J. Cai and X. Liu for discussions; and S. Xin, Y. Li, G. Ding, P. Wu and
S. Chen for technical assistance. Supported by the Ministry of Science and
Technology (2003CB515405 and 2005CB522406), the National Natural Science
Foundation of China (30021003, 30623003, 30625014, 30400230, 30430650 and
30571731), the Shanghai Municipal Commission for Science and Technology
(06ZR14098, 04JC14040, 04DZ14902 and PJ200500330), the Shanghai
Municipal Health Bureau (LJ06046), the Shanghai Leading Academic Discipline
Project (T206) and the Chinese Academy of Sciences (KSCX2-YW-R-56).
AUTHOR CONTRIBUTIONS
Y.S. designed and did the experiments and prepared the manuscript; Y.F. and
J.K. designed and did the experiments and analyzed the data; C.L. assisted with
cell purification, activation and culture; Z.L., Z.G. and C.L. provided the blood
samples from patients with multiple sclerosis; D.L. prepared the manuscript;
W.C. and J.Q. assisted with the induction of EAE; E.B. and L.Z. assisted with
cell purification, activation and culture; and J.Z.Z. and G.P. supervised all
studies and the preparation of the manuscript.
COMPETING INTERESTS STATEMENT
The authors declare no competing financial interests.
Published online at http://www.nature.com/natureimmunology/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions
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