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Departments of
*
Medicine III and
Microbiology,
Osaka University Medical School;
§
Biomolecular Engineering Research Institute; and
¶
Osaka University, Suita City, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, and IL-7 was inhibited,
suggesting that SSI-1 suppresses cytokine signaling in primary
lymphocytes. In addition, lck-SSI-1 transgenic mice
showed a reduction in the number of thymocytes as a result of the
developmental blocking during triple-negative stage. They also
exhibited a relative increase in the percentage of CD4+ T
cells, a reduction in the number of 
T cells, as well as the
spontaneous activation and increased apoptosis of peripheral T cells.
Thus, enforced expression of SSI-1 disturbs the development of
thymocytes and the homeostasis of peripheral T cells. All these
features of lck-SSI-1 transgenic mice strikingly
resemble the phenotype of mice lacking common -chain or Janus
kinase-3, suggesting that transgene-derived SSI-1 inhibits the
functions of common -chain-using cytokines. Taken together, these
results suggest that SSI-1 can also inhibit a wide variety of cytokines
in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been well established that cytokines play an essential role in T
cell development (3). In particular, cytokines that signal
through common -chain (c) are especially
essential for the development of T cells as well as B cells. Common
-chain is a shared component of receptors for IL-2, IL-4, IL-7,
IL-9, and IL-15 and is constitutively and specifically associated with
a member of JAK family, JAK3. The critical roles of
c and JAK3 in lymphoid development have been
demonstrated by the findings that mutations in either of these genes
cause SCID in humans. In addition, mice lacking
c or JAK3 show a similar immunodeficient
phenotype (4, 5). Thymocytes in these mice feature
developmental defects during the triple-negative (TN;
CD3-CD4-CD8-)
stage, and consequently their thymuses are strikingly small. Despite
their unresponsiveness to mitogenic stimuli in vitro, peripheral T
cells in these mice spontaneously express activation markers in vivo
and show an age-dependent expansion in the periphery. In addition,
these animals also exhibit a developmental defect in B cells, lack of
T cells, and NK cells (6, 7, 8, 9, 10).
These characteristics of
c-/- and
JAK3-/- mice are likely to be related to the
defective signal transduction of several
c-using cytokines such as IL-2, IL-7, and
IL-15. For example, a defect in early T and B cell development and
severe lymphocytopenia with lack of T cells are observed in mice
lacking IL-7 or IL-7R
(11, 12, 13). Spontaneous activation
and expansion of peripheral T cells are observed in mice lacking IL-2,
IL-2R, or IL-2Rß (14, 15, 16, 17). Lack of NK cells is
observed in mice deficient in IL-15R (18).
Recently, a new family of proteins has been identified and has been implicated in the regulation of cytokine signaling. These proteins are characterized by the presence of an SH2 domain and conserved C-terminal motifs (SC motifs; also referred to as the SOCS box) and variously called the STAT-induced STAT inhibitor (SSI) family (19, 20), the suppressor of cytokine signaling (SOCS) family (21, 22), or the cytokine-inducible SH2-containing protein (CIS) family (23, 24). Until now, eight members of this family have been identified, and their biological functions are currently being examined. Evidence to date suggests that at least three of them, CIS1, SSI-1/SOCS-1/JAB, and SSI-3/SOCS-3/CIS3, are involved in the negative feedback regulation in cytokine signaling (for a review, see Ref. 25).
Previous experiments in vitro have suggested that SSI-1, a member of this family, is a potent inhibitor of cytokine signaling. These experiments, using various cytokine-responsive cell lines, have shown that SSI-1 inhibits a wide variety of signal transduction of cytokines, such as IL-2, IL-3, IL-4, IL-6, leukemia inhibitory factor, IFNs, erythropoietin, thrombopoietin, growth hormone, and leptin (19, 21, 23, 26, 27, 28, 29). In addition, SSI-1 was shown in vitro to associate with all four members of JAKs (JAK1, JAK2, JAK3, and TYK2) and inhibit signaling by suppressing their kinase activities (19, 23, 30). These results suggest that SSI-1 can function as a general inhibitor of cytokine signaling by suppressing JAKs. On the other hand, interactions of SSI-1 with proteins other than JAKs have also been reported, such as its association with Tec and inhibition of IL-3 signaling (31) and its association with Grb-2 and Vav and inhibition of the proliferative function of stem cell factor (SCF) (32). Binding of SSI-1 to PYK2 (24) and insulin-like growth factor I receptor (33) in vitro was also reported, but their biological significance was not elucidated. These findings suggest that the functions of SSI-1 may not be limited to the inhibition of the JAK-STAT pathway.
To reveal the role of SSI-1 in vivo, SSI-1-/- mice have been generated (34, 35). Although the gross appearance of these mice is normal at birth, their growth is retarded with age. SSI-1-/- mice show progressive lymphocyte decrease due at least in part to accelerated apoptosis of lymphocytes. They also showed severe fatty degeneration in liver as well as less severe abnormalities in several organs, including the heart. In addition, all the animals died within 3 wk after birth. These results suggest that SSI-1 plays essential and nonredundant roles in normal neonatal development (34, 35).
More recently, two reports characterizing
SSI-1-/- mice were published (36, 37). One report indicated that SSI-1-/-
mice are hyper-responsive to exogenous as well as endogenous IFN-
(36). The other one indicated that SSI-1 is expressed in
developing thymocytes, and SSI-1 deficiency leads to the spontaneous
activation of T cells (37). It is likely that both IFN-
and T cells are critical to the disease and lethality of
SSI-1-/- mice, since the inactivation of
IFN- by introducing IFN- deficiency or the elimination of mature
lymphocytes by introducing recombinase-activating gene 2 deficiency can
prevent the phenotype of SSI-1-/- mice
(36, 37). Thus, SSI-1 appears to have an essential role in
the protection of mice from potentially toxic effects of IFN- and in
the regulation of lymphocyte functions. However, considering the
diverse inhibitory action of SSI-1 in vitro as well as the potential
toxicity of other cytokines, such as IL-4 and TNF-, whose
overexpression can induce lymphocyte depletion and neonatal lethality
in mice (38, 39), it remains to be elucidated whether
signaling of cytokines other than IFN- is perturbed in the absence
of SSI-1 in vivo.
Predominant expression of SSI-1 mRNA in lymphoid organs together with a
profound lymphocyte decrease in SSI-1-/- mice
suggest that SSI-1 has some essential roles in lymphocyte functions.
However, functions of SSI-1 in lymphocytes in vivo have hardly been
studied. For this purpose, we prepared lck-SSI-1 transgenic
mice by using the lck proximal promoter that strongly drives
transgene expression in T cell lineage, especially in thymocytes
(40, 41). Consistent with the findings obtained in vitro,
thymocytes overexpressing SSI-1 exhibited diminished activation of
STATs in response to cytokines such as IL-4, IL-6, IL-7, and IFN-.
Furthermore, lck-SSI-1 mice showed several defects in the
development of T cells, which can be attributed to the impaired
signaling through c and JAK3. These results
suggest that SSI-1 inhibits cytokine signaling in lymphocytes in vivo,
as has been shown in various cell lines in vitro, and the main action
of SSI-1 in lymphocytes is the suppression of the signaling of
cytokines, including not only IFN- but also other cytokines, such
as IL-7.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine SSI-1 cDNA was inserted into the BamHI site of the p1017 expression vector containing the lck proximal promoter (41). This construct was then injected into C57BL/6 blastocysts and implanted into pseudopregnant C57BL/6 mice. Founder mice were initially screened by PCR analysis of tail DNA using primers specific for lck proximal promoter (5'-ccagtcaggagcttgaatcc-3') and SSI-1 (5'-gcagctcgaaaaggcagtcg-3'). For four of the founder mice (lck-2, -8, -11, and -12), the expression of the SSI-1 transgene was confirmed by Northern and Western blot analysis. Animals from both lck-2 and lck-11 lines, which showed fairly strong expression of SSI-1 transgene, exhibited a similar phenotype, and those from the lck-11 line were used for the experiments described below. All mice were kept in specific pathogen-free facilities.
Cell preparation and cell counts
Single-cell suspensions were obtained from thymuses, spleens, and mesenteric lymph nodes after having been passed through mesh filters. Spleen cells were also treated with Ack buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM Na2EDTA) to lyse RBC. The total number of cells was determined by microscopic observation of trypan blue-stained cells using hemocytometers.
Western blot analysis
Thymocytes (2.5 x 106) were
stimulated with specified cytokines for 30 min or were left untreated.
The cytokine concentrations were 50 ng/ml for murine IL-4 (PeproTech,
London, U.K.), murine IL-6 (PharMingen, San Diego, CA), and murine IL-7
and murine IFN- (PeproTech). Cells were solubilized with ice-cold
lysis buffer containing 1% Nonidet P-40, 0.1% sodium deoxycholate,
0.1% SDS, 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF,
1 mM Na3VO4, and 5 µg/ml
aprotinin. Whole-cell lysates were separated on SDS-PAGE under reducing
conditions and transferred to nitrocellulose membranes. Membranes were
probed with following Abs: anti-SSI-1(1262B) (30),
anti-phospho-STAT1(Y701) (Upstate Biotechnology, Lake Placid, NY),
anti-phospho-STAT3(Tyr705), and
anti-phoshpo-STAT5(Tyr694) (New England
Biolabs, Beverly, MA) and then reprobed with Abs as follows:
anti-STAT1, anti-STAT3 (Transduction Laboratory, Lexington,
KY), and anti-STAT5b (this Ab also detects STAT5a; Santa Cruz
Biotechnology, Santa Cruz, CA).
Flow cytometric analysis
Cells were stained with the following Abs: FITC-, PE-, or
APC-conjugated anti-CD3e, -CD4, -CD8, -CD11, -CD25, -CD44, -CD62L,
and -CD69; TCRß; -TCR; pan-NK; and Gr-1 (all purchased from
PharMingen, San Diego, CA). Stained cells were analyzed on a
FACSCalibur (Becton Dickinson, San Jose, CA) using CellQuest software
(Becton Dickinson). Live lymphocytes were gated according to their
forward and side scatter profiles.
In the case of annexin V staining, freshly isolated cells were stained with anti-CD3e-PE and anti-B220-APC (PharMingen), and washed twice with PBS. Cells were further stained with annexin V-FITC (MBL, Nagoya, Japan) according to the manufacturer’s instructions. These cells were analyzed by flow cytometry as described above.
Thymocyte proliferation assay
For proliferation assays, 4 x 105 thymocytes/well in 96-well plates were cultured for 4 days in 100 µl of RPMI 1620 containing 5% FCS (Life Technologies, Tokyo, Japan), 2-ME (Nacalai Tesque, Kyoto, Japan), penicillin-G and streptomycin, and stimulated with 10 µg/ml plate-coated anti-CD3 (2C11; PharMingen) as well as with or without cytokines as indicated. The concentration used for cytokines were 20 ng/ml for murine IL-2, 20 ng/ml for murine IL-4, and 50 ng/ml for murine IL-7 (all purchased from PeproTech). Cell proliferation was determined by cell counting kit (Dojin Laboratories, Kumamoto, Japan) according to manufacturer’s instructions, and the results were shown as the absorbance at 450 nm (OD450).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify the in vivo function of SSI-1 in lymphocytes, we used
the lck proximal promoter to express SSI-1 in T cell lineage
of transgenic mice (Fig. 1
A).
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B, Western blot analysis showed highly expressed SSI-1
protein in lck-SSI-1 thymocytes, but not in splenocytes,
which is consistent with previous findings showing that the transgene
expression driven by the lck proximal promoter is strong in
thymocytes, but declines in mature T cells (40). SSI-1 inhibits the activation of STATs in primary lymphocytes
Using lck-SSI-1 thymocytes, we first checked the
function of SSI-1 in cytokine signaling of lymphocytes. When thymocytes
from lck-SSI-1 mice and their littermates were stimulated
with several cytokines, tyrosine phosphorylation of STATs in response
to IL-4, IL-6, IL-7, or IFN- was inhibited in lck-SSI-1
thymocytes (Fig. 1C and data not shown). Thus, SSI-1 was
found to suppress cytokine signaling in primary lymphocytes.
lck-SSI-1 mice show a decrease in the number of T cells
lck-SSI-1 mice appeared healthy and normal, except for
their markedly small thymuses and slightly atrophic spleens. The
cellularity of thymuses and spleens were 6 and 1.5 times fewer than
those of the wild-type littermates, respectively (Fig. 1D).
Furthermore, the number of splenic T cells, estimated on the basis of
the percentage of CD3-positive splenocytes, was reduced by 10-fold,
even more severely reduced than the thymocytes. Thus, enforced
expression of SSI-1 in T cell lineage results in a selective decrease
in T cells.
Early thymocyte development is impaired in lck-SSI-1 mice
To characterize the reduction in the number of thymocytes,
we analyzed lck-SSI-1 thymocytes by flow cytometry. The
expression profiles of CD4 and CD8 were normal in lck-SSI-1 mice (Fig. 2A) except for a slight
increase in the CD4:CD8 ratio (Fig. 2B). This indicates that
the absolute number of lck-SSI-1 thymocytes had already been
reduced at the CD4-CD8-
stage. We therefore focused on immature thymocytes gating on the TN
(CD3-CD4-CD8-)
fraction. TN thymocytes can be divided into four subsets according to
their expression of CD25 and CD44, and their maturation sequence is as
follows: CD44+CD25-
(pro-T1), CD44+CD25+
(pro-T2), CD44-CD25+
(pro-T3), and CD44-CD25-
(pro-T4) (3). In lck-SSI-1 TN thymocytes, a
decrease in the percentages of pro-T3 and pro-T4 thymocytes was seen as
well as an increase in the percentages of earlier subsets, namely
pro-T1 and pro-T2 cells (Fig. 2C). This finding suggests
that early thymocyte development is partially blocked around the pro-T2
to pro-T3 transition in lck-SSI-1 mice. In addition, the
percentage of T cells, which are known to be generated from
pro-T2 cells (3), was reduced in lck-SSI-1
thymocytes, although the percentage of ß T cells was comparable to
that in control littermates (Fig. 2D).
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We next analyzed the peripheral lymphocytes of
lck-SSI-1 mice. As shown in Fig. 3, FACS analysis of lck-SSI-1
splenocytes and lymph node cells revealed a severe decrease in
peripheral T cells. In addition, peripheral T cells of
lck-SSI-1 mice showed an apparently higher CD4:CD8 ratio
than control cells (Fig. 3D). On the other hand, the
development of B cells in lck-SSI-1 mice was normal, as
judged by the expression of CD23, IgM, and IgD (data not shown), and
their number was also normal, which is consistent with T cell-specific
expression of SSI-1 transgene under the control of the lck
proximal promoter.
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, increased proportions of
both CD4 and CD8 splenic T cells from lck-SSI-1 mice showed
an up-regulation of CD44 and CD69 (data not shown) and a
down-regulation of CD62L, which are characteristics of activated/memory
T cells. These results suggest that peripheral T cells in
lck-SSI-1 mice are activated spontaneously in vivo.
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Despite the enhanced activation of peripheral T cells, the number
of T cells in the periphery was severely reduced, as shown in Fig. 1D. This decrease in peripheral T cells was also observed in
older (1 year of age) lck-SSI-1 mice. To examine the cause
of this reduction, we analyzed the apoptosis of lymphocytes by staining
freshly prepared splenocytes with annexin V, which detects early
apoptotic cells by binding to phosphatidylserine on the cell surface.
As shown in Fig. 5, annexin V-positive
CD3+ T cells were increased in
lck-SSI-1 splenocytes. This increase seemed to be specific
to T cells, as the percentage of annexin V-positive B cells was not
increased in lck-SSI-1 mice. Thus, lck-SSI-1 mice
show an increase in apoptosis of peripheral T cells.
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). Consistent with the
increase in the percentage of activated T cells, the expression of Fas
was slightly up-regulated in lck-SSI-1 splenic T cells. In
addition, we found that FasL expression in lck-SSI-1 T cells
was up-regulated compared with that in littermates. These results
suggest that the increased apoptosis of lck-SSI-1 T cells is
partly mediated by the Fas-FasL systems.
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c or JAK3
Previous reports using gene-targeting technique have revealed a
wide variety of molecules that play essential roles in T cell
development. Among them, the phenotype of lck-SSI-1 mice
strikingly resembles that of T cells lacking c
or JAK3. Both c-/- and
JAK3-/- mice show an indistinguishable
phenotype in T cells that is characterized by a defective thymocyte
development with a fairly normal expression of CD4 and CD8 (except for
a slight increase in the CD4:CD8 ratio), a partial developmental block
around the pro-T2 and pro-T3 transition, a lack of T cells and
spontaneous activation, as well as increased apoptosis of peripheral T
cells (6, 7, 8, 9, 10, 43, 44, 45, 46, 47, 48, 49). All these characteristics was also
seen in lck-SSI-1 mice, suggesting that cytokines that
signal through c and JAK3 were inhibited in T
cells of lck-SSI-1 mice. This idea is further supported by
the results shown in Fig. 7, demonstrating that lck-SSI-1 thymocytes were hyporesponsive
not only to IL-7, but also to other c- and
JAK3-using cytokines, such as IL-2 and IL-4.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c or JAK3
In this study we demonstrated that enforced expression of SSI-1 in
the T cell lineage results in impaired development of thymocytes as
well as enhanced activation and apoptosis of peripheral T cells.
Furthermore, this phenotype of lck-SSI-1 mice is quite
similar to that of mice lacking c or JAK3,
strongly suggesting that SSI-1 can inhibit in vivo the signaling of
cytokines that signal through c and JAK3. This
supposition is supported by the finding that lck-SSI-1
thymocytes showed hyporesponsiveness to c- and
JAK3-using cytokines such as IL-2, IL-4, and IL-7. (Figs. 1C
and 7) and is in agreement with the previous findings in vitro that
SSI-1 inhibits various cytokine signaling by suppressing JAKs
(25).
Recently, SSI-1 was shown to have an inhibitory role in signaling
through c-Kit, a receptor for SCF (32). SCF signaling is
also known to play an essential role in early thymocyte development
(3). However, the phenotype of lck-SSI-1 mice
is not attributable to the defect in this SCF/c-Kit pathway, since
c-Kit is expressed only in pro-T1 and pro-T2 thymocytes, and mice
encoding mutant c-Kit (w/w) showed a severe reduction, not a relative
increase, in these most immature thymocytes (50). Thus, as
denoted above, it is likely that developmental defects in
lck-SSI-1 thymocytes are essentially attributable to the
suppression of c-JAK3 signaling pathways.
Further studies are required, however, to rule out the possibility that
defects in other signaling pathways were partly implicated in the
phenotype of lck-SSI-1 mice.
Phenotypic difference between lck-SSI-1 mice and
mice lacking c or JAK3
As well as the similarities, there were several phenotypic
differences between lck-SSI-1 mice and mice lacking
c or JAK3 (45, 46, 47, 48, 49). In thymus,
the decrease in the number of thymocytes of lck-SSI-1 mice
(
6-fold) was less dramatic than that observed in these knockouts
(>10-fold). In addition, NK cells, which were absent in
c-/- and
JAK3-/- mice, were not reduced in
lck-SSI-1 mice. In the periphery, although
lck-SSI-1 T cells showed activated/memory phenotype,
age-dependent accumulation of CD4+ T cells, which
was commonly observed in
c-/- and
JAK3-/- mice, was not observed in
lck-SSI-1 mice.
All these differences appear to be explained by the characteristics of
the lck proximal promoter. Previous reports suggested that
the lck proximal promoter is not efficient for the transgene
expression in most immature thymocytes, such as common lymphoid
progenitors and pro-T1 cells (51). Therefore, it is
conceivable that thymocytes at their most immature stage in
lck-SSI-1 mice are less affected by SSI-1 transgene
expression, resulting in a less severe reduction in the number of
lck-SSI-1 mice. This hypothesis also explains the presence
of NK cells in lck-SSI-1 mice because NK cells are known to
develop from most immature thymocytes. On the other hand, it is known
that the expression driven by the lck proximal promoter
declines in peripheral T cells (40). In line with this,
the expression of the SSI-1 transgene in the periphery was not detected
by Northern (data not shown) or Western (Fig. 1B) analysis,
but only by RT-PCR analysis (data not shown). Thus, it is highly
possible that lck-SSI-1 T cells gradually lose transgene
expression after emigration from the thymus and recover their ability
to respond to cytokines in the periphery. In the case of
c-/- and
JAK3-/- mice, lack of IL-2 signaling appears to
cause the peripheral expansion of activated T cells, since IL-2 is
critical in sensitizing T cells to AICD (42). Indeed, it
was previously reported that superantigen-induced deletion of
peripheral T cells is impaired in
c-/- mice as a result of
defective FasL expression in activated T cells (52). In
this point of view, it seems significant that lck-SSI-1
splenic T cells showed increased expression of Fas and FasL (Fig. 6).
Thus, in lck-SSI-1 mice, although emigrants from thymus
became activated in the periphery, these T cells appear to be deleted
by Fas-mediated AICD after they become responsive to cytokines such
as IL-2.
SSI-1 inhibits downstream signaling of JAKs
Previous in vitro analyses of CIS1, the first member of the SSI family, have suggested that CIS1 inhibits the function of STAT5 by masking the STAT5 binding site of cytokine receptors. A recent report has shown that CIS1 transgenic mice exhibit a phenotype similar to that of mice lacking STAT5a/b, suggesting that CIS1 specifically inhibits STAT5 also in vivo (53). In contrast, the phenotype of lck-SSI-1 mice is not attributable only to the inhibition of a single member of STATs, since none of the known knockout mice of STATs (including STAT1, STAT3, STAT4, STAT5a/b, and STAT6) shows an apparent defect in T cell development (54, 55, 56, 57, 58, 59, 60, 61, 62). This is in line with the previous findings in vitro that SSI-1 associates not with cytokine receptors but with JAKs and inhibits their kinase activity.
Judging from the observations that SSI-1 inhibits JAKs in vitro, close
similarity between lck-SSI-1 mice and mice lacking
c or JAK3 suggests that the function of JAK3
is inhibited in lck-SSI-1 mice. It should, however, be noted
that SSI-1 is not a specific inhibitor of JAK3, because the
phosphorylation of STATs in response to IL-6 and IFN-, which require
neither JAK3 nor c for their signal
transduction, was inhibited in lck-SSI-1 thymocytes. In
addition, we cannot rule out the possibility that the phenotype of
lck-SSI-1 mice may be attributed entirely to the suppression
of JAK1, since JAK1 constitutively associates with several cytokine
receptor systems, including IL-2R, IL-4R, and IL-7R, and
JAK1-/- mice, although less defined than
JAK3-/- mice, also exhibit a defect in early
lymphoid development (63). This hypothesis can also
explain the finding that the phosphorylation of STATs in response to
IL-6 and IFN- was inhibited in lck-SSI-1 thymocytes.
Thus, although the phenotype of lck-SSI-1 mice can be
attributed to the inhibition of JAKs, more information on
JAK1-/- mice and further studies are required
to specify the precise targets of SSI-1’s action in
lck-SSI-1 mice.
Functions of SSI-1 in lymphocytes
Lines of previous evidence suggest that SSI-1 is important for the regulation of lymphocyte functions. SSI-1 mRNA is predominantly expressed in lymphoid organs such as thymus and spleen (19, 21), and T cells as well as B cells themselves express SSI-1 in vivo (35). However, the function of SSI-1 in primary lymphocytes has not been fully studied to date. We have shown here that SSI-1 can efficiently inhibit cytokine signaling in primary lymphocytes also. Moreover, as the phenotype of lck-SSI-1 mice can essentially be attributed to the suppression of JAKs, the inhibition of cytokine signaling appears to be the most essential function of SSI-1 in lymphocytes.
Recently, it was reported that SSI-1 deficiency leads to a reduction in the number of lymphocytes (34, 35) and a spontaneous activation of T cells (37). Interestingly, as we showed here, enforced expression of SSI-1 in T cells also ended in T cell reduction and activation. These results indicate that inadequate expression of SSI-1 in T cells, both too little and too much, results in a similar perturbation of their development and homeostasis. In particular, loss of SSI-1 in lymphocytes appears to be critical, since mice reconstituted with SSI-1-/- lymphocytes exhibit lethality similar to that in SSI-1-/- mice (37). Thus, tightly regulated expression of SSI-1 in T cells is likely to be required to balance the effect of cytokines in vivo.
Functions of SSI-1 in cytokine signaling
It has been shown that SSI-1 in vitro suppresses a wide variety of
cytokine signaling by inhibiting JAKs. Subsequent generation of
SSI-1-/- mice revealed that SSI-1 deficiency
leads to a complex and fatal disease characterized by progressive
lymphocyte depletion, fatty degeneration of liver, and neonatal
lethality within 3 wk after birth (34, 35). Surprisingly,
despite the diverse action of SSI-1 in vitro, recent reports have shown
that introducing IFN- deficiency can essentially prevent the disease
and lethality of SSI-1-/- mice (36, 37). Thus, these results suggest the possibility that SSI-1 is a
specific inhibitor of IFN-.
However, lines of evidence argue against this supposition. As described
earlier, several groups have shown in various experiments in vitro that
SSI-1 binds to JAKs and thereby inhibits their kinase activity
(30, 64, 65). IFN--specific action cannot be explained
by this well-established inhibitory mechanism of SSI-1, since JAK1 and
JAK2, which function as downstream kinases of IFN- signaling, have
nonredundant roles for signaling of several cytokines other than
IFN- (63, 66, 67). Therefore, inhibiting JAK1 or JAK2
results in the suppression of not only IFN-, but other cytokines
also, such as IL-6 or erythropoietin. In addition to this, our present
study of transgenic mice clearly suggests that SSI-1 in vivo can
inhibit the signaling of several cytokines including not only IFN-
but also others, such as IL-7.
Although our transgenic model may lead to the overestimation of
SSI-1’s functions, several findings in
SSI-1-/- mice suggest that SSI-1 is not
specific to IFN- signaling. It was previously demonstrated that
SSI-1-/- thymocytes proliferate more vigorously
than SSI-1+/+ thymocytes in response to
anti-CD3 plus IL-2 or IL-4. In line with this,
SSI-1-/- thymocytes showed markedly sustained
tyrosine phosphorylation of STAT6 in response to IL-4
(34). Recently, it was shown that
SSI-1-/- splenocytes exhibit enhanced
proliferation in response to IL-2 alone, suggesting their
hyper-responsiveness to IL-2 (37). This
hyper-responsiveness to cytokines such as IL-2 may be a reason for the
spontaneous activation of SSI-1-/- T cells in
vivo. In addition, interestingly, our preliminary data showed that
SSI-1/STAT6 double-knockout mice were partially rescued from the
disease and the neonatal lethality seen in mice lacking SSI-1 alone
(our unpublished observations). As STAT6 is an essential molecule for
IL-4 and IL-13 signaling (60, 61, 62, 68), this result
suggests that IL-4, IL-13, or both have an additional or synergistic
effect on the toxicity of IFN- in SSI-1-/-
mice. This result also raises the possibility that the complex action
of other cytokines, which might be masked by the devastating toxicity
of IFN-, is partly implicated in the phenotype of
SSI-1-/- mice. Taken together, we propose here
that SSI-1 has the potential to inhibit the signaling of multiple
cytokines in vivo. Further studies are currently underway to elucidate
the diversity of SSI-1 function in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tadamitsu Kishimoto, Osaka University, 1-1 Yamada-oka, Suita City, Osaka 565-0871, Japan.
3 Abbreviations used in this paper: JAK, Janus kinase; AICD, activation-induced cell death; APC, allophycocyanin; CIS, cytokine-inducible SH2-containing protein; CD62L, CD62 ligand; c, common -chain; FasL, Fas ligand; SCF, stem cell factor; SH2, Src homology domain 2; SOCS, suppressor of cytokine signaling; SSI, STAT-induced STAT inhibitor; TN, triple negative.
Received for publication February 29, 2000. Accepted for publication May 30, 2000.
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References |
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chain. Proc. Natl. Acad. Sci. USA 92:377. chain. Immunity 2:223.[Medline]
T cell development and early thymocyte maturation in IL-7-/- mice. J. Immunol. 157:2366.[Abstract]
chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3:521.[Medline]
signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell 98:597.[Medline]
-chain of cytokine receptors regulates intrathymic T cell development at multiple stages. J. Immunol. 158:2592.[Abstract]
chain and the pre-T cell receptor provide independent but critically overlapping signals in early /ß T cell development. J. Exp. Med. 189:563.ß and thymocyte development. J. Immunol. 162:1448. chain plays an essential role in regulating lymphoid homeostasis. J. Exp. Med. 185:189.ß T cell development in mice deficient in the common cytokine receptor -chain: the requirement for Bcl-2 differs depending on the TCR/MHC affinity. J. Immunol. 162:782.-chain: defective Fas ligand expression in -chain-deficient mice. J. Immunol. 159:4737.[Abstract]
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